Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza hemagglutinin

ABSTRACT

The invention features a novel influenza antibody that specifically binds to influenza hemagglutinin and reduces or inhibits hemagglutinin binding to sialic acid. The invention also provides methods, compositions, and kits featuring the novel antibody and its use in preventing or treating influenza infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.14/171,322, filed Feb. 3, 2014, which is a Continuation-in-Part filingof PCT/US2012/49573, filed on Aug. 3, 2012, which claims the benefit ofU.S. Provisonal Patent Application No. 61/514,662, filed Aug. 3, 2011,the contents of all of which are incorporated herein by reference intheir entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This work was supported by grant nos. U19 AI067854 and P01GM62580 fromthe National Institutes of Health. The Government has certain rights inthis invention.

BACKGROUND OF THE INVENTION

The well-known seasonal drift of influenza virus antigenicity accountsfor the absence of long-term immune protection in previously infectedindividuals. The hemagglutinin (HA), a trimeric surface glycoproteinthat binds the viral receptor and promotes fusion and penetration fromlow-pH endosomes, is the principal surface antigen on influenza virions.HA presents conserved as well as variable epitopes, but neutralizingantibodies against the latter dominate the response to immunization andinfection.

Accordingly, there is a need for developing broadly neutralizingtherapeutics that can effectively treat or prevent drifted strains ofinfluenza.

SEQUENCE SUBMISSION

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is entitled“048218-519NO1US_Seq_Listing_03FEB2014”, was created on Feb. 3, 2014,with a file size of 85 KB. The information in the electronic format ofthe Sequence Listing is part of the present application and isincorporated herein by reference in its entirety.

SUMMARY OF THE INVENTION

As described below, the present invention is based upon the discovery ofnovel antibodies that broadly neutralize influenza antigenic variants.The invention features compositions and kits containing the novelantibodies, as well as methods for using these novel therapeuticmolecules to treat or prevent influenza viral infection.

In aspects, the invention provides isolated anti-influenza antibody orantibody fragment that specifically binds to an epitope of an influenzahemagglutinin (HA). Binding of the antibody or antibody fragment to theinfluenza HA epitope reduces or inhibits influenza HA binding to sialicacid.

The present invention also provides in other aspects an isolatedanti-influenza antibody or antibody fragment that specifically binds toa sialic acid binding domain of a surface antigen of influenza virus. Inone embodiment, the surface antigen of influenza virus is HA.

In embodiments, the epitope of influenza HA comprises a sialic-acidbinding domain.

In embodiments, the HA is H1 HA, H2 HA, H3 HA, or H5 HA (an HA from ahuman adapted H5 strain).

In related embodiments, the antibody contacts one or more residues ininfluenza HA epitope comprising residue 158; residues 158-160; 135-136,190-195, and 226; 222, 225, and 227; and residues 187 and 189 where thenumbering refers to the one used in structures such as that forA/Solomon Islands/3/2006 (Protein Data Bank accession number 3SM5).

In related embodiments, the influenza HA epitope comprises the aminoacids set forth in any one of SEQ ID NOs:17-44.

In related embodiments, the influenza HA epitope comprises the CH65-CH67binding residues in any one of SEQ ID NOs:17-44 (e.g., the CH65-CH67binding residues identified in FIG. 15).

In other embodiments, the anti-influenza antibody or antibody fragmentcontacts one or more residues in the sialic acid binding pocket of theHA epitope selected from the group consisting of residues: 134-136,190-195 and 226. In another embodiment, the antibody or antibodyfragment further contacts one or more residues in the HA epitopeselected from the group consisting of residues: 158, 158-160, 135-136,190-195 and 226, and 187 and 189, where the numbering refers to the oneused in structures such as that for A/Solomon Islands/3/2006 (ProteinData Bank accession number 3SM5).

In another embodiment, the antibody CDR H1 region contacts residue 158of the HA epitope; the CDR H2 region contacts residues 158-160 of the HAepitope; the CDR H3 region contacts residues 135-136, 190-194 and 226 ofthe HA epitope; the CDR L1 region contacts residues 222, 225 and 227 ofthe HA epitope; or the CDR L3 region contacts residues 187 and 198 ofthe HA epitope.

In embodiments, the anti-influenza antibody or antibody fragmentcomprises a variable heavy (V_(H)) chain, and wherein the V_(H) chaincomprises an amino acid sequence set forth in SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, or SEQ ID NO: 12.

In embodiments, the anti-influenza antibody or antibody fragmentcomprises one or more heavy chain CDR regions present in a variableheavy (V_(H)) chain amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, or SEQ ID NO: 12. In related embodiments, the one or moreheavy chain CDR regions comprises a CDR3 region present in the variableheavy (V_(H)) chain amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, or SEQ ID NO: 12.

In embodiments, the anti-influenza antibody or antibody fragmentcomprises a variable light (V_(L)) chain, and wherein the V_(L) chaincomprises an amino acid sequence set forth in SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, or SEQ ID NO: 16.

In embodiments, the anti-influenza antibody or antibody fragmentcomprises one or more light chain CDR regions present in a variablelight (V_(L)) chain amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14,SEQ ID NO: 15, or SEQ ID NO: 16. In related embodiments, the one or morelight chain CDR regions comprises a CDR3 region present in the variableheavy (V_(L)) chain amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14,SEQ ID NO: 15, or SEQ ID NO: 16.

In embodiments, the anti-influenza antibody or antibody fragmentcomprises i) a variable heavy (V_(H)) chain amino comprising an aminoacid sequence set forth in SEQ ID NO: 10, and ii) a variable light(V_(L)) chain comprising an amino acid sequence set forth in SEQ ID NO:14.

In embodiments, the anti-influenza antibody or antibody fragmentcomprises a variable heavy (V_(H)) chain, wherein the CDR3 region of theV_(H) chain comprises Arg104, Ser105, Val106, Asp107, Tyr109, Tyr110,Tyr112, or a combination thereof.

In embodiments, the anti-influenza antibody is a monoclonal antibody orantibody fragment thereof.

In embodiments, the anti-influenza antibody is a humanized antibody.

In embodiments, the antibody fragment is an Fab fragment, an Fab′fragment, an Fd fragment, a Fd′ fragment, an Fv fragment, a dAbfragment, an F(ab′)2 fragment, a single chain fragment, a diabody, or alinear antibody.

In embodiments, the anti-influenza antibody or antibody fragment furthercomprises an agent conjugated to the anti-influenza antibody or antibodyfragment thereof. In related embodiments, the agent conjugated to theantibody or antibody fragment thereof is a therapeutic agent ordetectable label.

The therapeutic agent can be any therapeutic agent suitable for use withthe novel antibodies. Such agents are well known in the art and includesmall molecules, nanoparticles, polypeptides, radioisotopes, inhibitorynucleic acids, and the like. In embodiments, the therapeutic agent is anantiviral agent or a toxin. In embodiments, the therapeutic agent is ansiRNA, shRNA, or antisense nucleic acid molecule that reduces influenzavirus production.

The detectable label can be any detectable label suitable for use withthe novel antibodies. Such labels are well known in the art and includelabels that are detected by spectroscopic, photochemical, biochemical,immunochemical, physical, or chemical means. In embodiments, thedetectable label is an enzyme, a fluorescent molecule, a particle label,an electron-dense reagent, a radiolabel, a microbubble, biotin,digoxigenin, or a hapten or a protein that has been made detectable.

In aspects, the invention provides pharmaceutical compositionscontaining at least one of the anti-influenza antibody or antibodyfragments described herein. In embodiments, the pharmaceuticalcompositions contain a pharmaceutically acceptable carrier, diluent, orexcipient.

In aspects, the invention provides isolated polynucleotides encoding ananti-influenza antibody or antibody fragments described herein. Inrelated aspects, the invention provides expression vectors comprisingsuch an isolated polynucleotide. In further related aspects, theinvention provides host cells comprising such an expression vector.

In aspects, the invention provides methods for treating or preventing aninfluenza virus infection in a subject in need thereof. The methodsinvolve administering to the subject an effective amount of ananti-influenza antibody or antibody fragment described herein, or apharmaceutical composition containing the antibody or antibody fragment.The methods treat or prevent influenza virus infection in the subject,including reducing or alleviating symptoms associated with infection.

In aspects, the invention provides methods for neutralizing an influenzavirus in a subject in need thereof. The methods involve administering tothe subject an effective amount of an anti-influenza antibody orantibody fragment described herein, or a pharmaceutical compositioncontaining the antibody or antibody fragment. The methods neutralize theinfluenza virus in the subject, thereby treating or prevent influenzavirus infection in the subject, including reducing or alleviatingsymptoms associated with infection.

In aspects, the invention provides methods for establishment ofinfluenza virus infection in a subject in need thereof. The methodsinvolve administering to the subject an effective amount of ananti-influenza antibody or antibody fragment described herein, or apharmaceutical composition containing the antibody or antibody fragment.The methods inhibit establishment of influenza virus infection in thesubject, thereby preventing symptoms associated with infection.

In aspects, the invention provides methods for inhibiting disseminationof influenza virus infection in a subject in need thereof. The methodsinvolve administering to the subject an effective amount of ananti-influenza antibody or antibody fragment described herein, or apharmaceutical composition containing the antibody or antibody fragment.The methods inhibit dissemination of influenza virus infection in thesubject, thereby reducing or alleviating symptoms associated withinfection.

In aspects, the invention provides methods for inhibiting influenzavirus entry into a cell in a subject. The methods involve administeringto the subject an effective amount of an anti-influenza antibody orantibody fragment described herein, or a pharmaceutical compositioncontaining the antibody or antibody fragment. The methods inhibitinfluenza virus entry into a cell in the subject, thereby preventingsymptoms associated with infection or reducing or alleviating symptomsassociated with infection.

In aspects, the invention provides methods for inhibiting influenzavirus entry into a cell. The methods involve contacting a cell having orat risk of developing influenza virus infection with an anti-influenzaantibody or antibody fragment described herein, or a pharmaceuticalcomposition containing the antibody or antibody fragment. The methodsinhibit influenza virus entry into the cell.

In any of the above aspects, the subject has or is at risk of developingan influenza infection. In related embodiments, the subject is a mammal(e.g., human). In related embodiments, the subject is susceptible toviral infection (e.g., a pregnant female, a young subject or an infantsubject, an elderly subject).

In any of the above aspects and embodiments, the anti-influenza antibodyor antibody fragment, or the pharmaceutical composition is administeredby intramuscular injection, intravenous injection, subcutaneousinjection, or inhalation.

In aspects, the invention provides kits for treating or preventinginfluenza virus infection; kits for neutralizing influenza virus; kitsfor inhibiting establishment of influenza virus infection; kits forinhibiting dissemination of influenza virus infection; and kits forinhibiting influenza virus entry into a cell.

In embodiments, the kits contain an anti-influenza antibody or antibodyfragment described herein.

In embodiments, the kits also contain a therapeutic agent. In relatedembodiments, the therapeutic agent inhibits influenza infection.

In embodiments, the kits also contain directions for using the kits inany of the methods described herein.

In any of the above embodiments, the influenza can be H1N1, H2N2, H3N2,or a human adapted H5 influenza strain (i.e., an H5 influenza that hasacquired human-receptor specificity; see FIG. 14D for exemplarystrains).

Additional objects and advantages of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the invention. Theobjects and advantages of the invention will be realized and attained bymeans of the elements and combinations disclosed herein, including thosepointed out in the appended claims. It is to be understood that both theforegoing general description and the following detailed description areexemplary and explanatory only and are not restrictive of the inventionas claimed. The accompanying drawings, which are incorporated herein andconstitute a part of this specification, illustrate several embodimentsof the invention and, together with the description, serve to explainthe principles of the invention.

Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

As used herein, the singular forms “a”, “an”, and “the” include pluralforms unless the context clearly dictates otherwise. Thus, for example,reference to “an influenza antibody” includes reference to more than oneinfluenza antibody.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive.

As used herein, the terms “comprises,” “comprising,” “containing,”“having” and the like can have the meaning ascribed to them in U.S.Patent law and can mean “includes,” “including,” and the like;“consisting essentially of” or “consists essentially” likewise has themeaning ascribed in U.S. Patent law and the term is open-ended, allowingfor the presence of more than that which is recited so long as basic ornovel characteristics of that which is recited is not changed by thepresence of more than that which is recited, but excludes prior artembodiments.

The term “antibody” means an immunoglobulin molecule that recognizes andspecifically binds to a target, such as a protein, polypeptide, peptide,carbohydrate, polynucleotide, lipid, or combinations of the foregoingthrough at least one antigen recognition site within the variable regionof the immunoglobulin molecule. As used herein, the term “antibody”encompasses intact polyclonal antibodies, intact monoclonal antibodies,antibody fragments (such as Fab, Fab′, F(ab′)2, and Fv fragments),single chain Fv (scFv) mutants, multispecific antibodies such asbispecific antibodies generated from at least two intact antibodies,chimeric antibodies, humanized antibodies, human antibodies, fusionproteins comprising an antigen determination portion of an antibody, andany other modified immunoglobulin molecule comprising an antigenrecognition site so long as the antibodies exhibit the desiredbiological activity. An antibody can be of any the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively. The different classes ofimmunoglobulins have different and well known subunit structures andthree-dimensional configurations. Antibodies can be naked or conjugatedto other molecules such as toxins, radioisotopes, and the like.

The basic four-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains. An IgM antibody consists of 5 of the basic heterotetramer unitalong with an additional polypeptide called J chain, and thereforecontains 10 antigen binding sites, while secreted IgA antibodies canpolymerize to form polyvalent assemblages comprising 2-5 of the basic4-chain units along with J chain. In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to an H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(V_(H)) followed by three constant domains (C_(H)) for each of the α andγ chains and four C_(H) domains for μ and ε isotypes. Each L chain hasat the N-terminus, a variable domain (V_(L)) followed by a constantdomain (C_(L)) at its other end. The V_(L) is aligned with the V_(H) andthe C_(L) is aligned with the first constant domain of the heavy chain(CH1). Particular amino acid residues are believed to form an interfacebetween the light chain and heavy chain variable domains. The pairing ofa V_(H) and V_(L) together forms a single antigen-binding site. For thestructure and properties of the different classes of antibodies, see,e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, AbbaI. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk,Conn., 1994, page 71, and Chapter 6.

An “isolated antibody” is one that has been separated and/or recoveredfrom a component of its natural environment. Contaminant components ofits natural environment are materials that would interfere withdiagnostic or therapeutic uses for the antibody, and may includeenzymes, hormones, and other proteinaceous or nonproteinaceous solutes.In embodiments, the antibody is purified: (1) to 80%, 85%, 90%, 95%, 99%or more by weight of antibody as determined by the Lowry method; (2) toa degree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator; or (3)to homogeneity by SDS-PAGE under reducing or non-reducing conditionsusing Coomassie blue, silver stain, and the like. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.In embodiments, an isolated antibody will be prepared by at least onepurification step.

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limited toFab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, single chainantibodies, and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire L chain along with the variable regiondomain of the H chain (V_(H)), and the first constant domain of oneheavy chain (CH1). Each Fab fragment is monovalent with respect toantigen binding, i.e., it has a single antigen-binding site. Pepsintreatment of an antibody yields a single large F(ab′)2 fragment thatroughly corresponds to two disulfide linked Fab fragments havingdivalent antigen-binding activity and is still capable of cross-linkingantigen. Fab′ fragments differ from Fab fragments by having additionalfew residues at the carboxy terminus of the CH1 domain including one ormore cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)2 antibody fragmentsoriginally were produced as pairs of Fab′ fragments that have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The “Fc” fragment comprises the carboxy-terminal portions of both Hchains held together by disulfides. The effector functions of antibodiesare determined by sequences in the Fc region, which region is also thepart recognized by Fc receptors (FcR) found on certain types of cells.

An “Fv antibody” refers to the minimal antibody fragment that contains acomplete antigen-recognition and -binding site either as two-chains, inwhich one heavy and one light chain variable domain form a non-covalentdimer, or as a single-chain (scFv or sFv), in which one heavy and onelight chain variable domain are covalently linked by a flexible peptidelinker so that the two chains associate in a similar dimeric structure.In this configuration the complementary determining regions (CDRs) ofeach variable domain interact to define the antigen-binding specificityof the Fv dimer. Alternatively a single variable domain (or half of anFv) can be used to recognize and bind antigen, although generally withlower affinity.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10 residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,resulting in a bivalent fragment, i.e., fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

A “monoclonal antibody” refers to homogenous antibody populationinvolved in the highly specific recognition and binding of a singleantigenic determinant, or epitope. This is in contrast to polyclonalantibodies that typically include different antibodies directed againstdifferent antigenic determinants. The term “monoclonal antibody”encompasses both intact and full-length monoclonal antibodies as well asantibody fragments (such as Fab, Fab′, F(ab′)2, Fv), single chain (scFv)mutants, fusion proteins comprising an antibody portion, and any othermodified immunoglobulin molecule comprising an antigen recognition site.Furthermore, “monoclonal antibody” refers to such antibodies made in anynumber of manners including but not limited to by hybridoma, phageselection, recombinant expression, and transgenic animals.

The term “humanized antibody” refers to forms of non-human (e.g.,murine) antibodies that are specific immunoglobulin chains, chimericimmunoglobulins, or fragments thereof that contain minimal non-human(e.g., murine) sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues from the complementary determiningregion (CDR) are replaced by residues from the CDR of a non-humanspecies (e.g. mouse, rat, rabbit, hamster, and the like) that have thedesired specificity, affinity, and capability (Jones et al., 1986,Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327;Verhoeyen et al., 1988, Science, 239:1534-1536). In some instances, theFv framework region (FR) residues of a human immunoglobulin are replacedwith the corresponding residues in an antibody from a non-human speciesthat has the desired specificity, affinity, and capability. Thehumanized antibody can be further modified by the substitution ofadditional residue either in the Fv framework region and/or within thereplaced non-human residues to refine and optimize antibody specificity,affinity, and/or capability. In general, the humanized antibody willcomprise substantially all of at least one, and typically two or three,variable domains containing all or substantially all of the CDR regionsthat correspond to the non-human immunoglobulin whereas all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody can also comprise at least aportion of an immunoglobulin constant region or domain (Fc), typicallythat of a human immunoglobulin. Examples of methods used to generatehumanized antibodies are described in U.S. Pat. No. 5,225,539.

The term “human antibody” means an antibody produced by a human or anantibody having an amino acid sequence corresponding to an antibodyproduced by a human made using any technique known in the art. Thisdefinition of a human antibody includes intact or full-lengthantibodies, fragments thereof, and/or antibodies comprising at least onehuman heavy and/or light chain polypeptide such as, for example, anantibody comprising murine light chain and human heavy chainpolypeptides.

“Hybrid antibodies” are immunoglobulin molecules in which pairs of heavyand light chains from antibodies with different antigenic determinantregions are assembled together so that two different epitopes or twodifferent antigens can be recognized and bound by the resultingtetramer.

The term “chimeric antibodies” refers to antibodies wherein the aminoacid sequence of the immunoglobulin molecule is derived from two or morespecies. Typically, the variable region of both light and heavy chainscorresponds to the variable region of antibodies derived from onespecies of mammals (e.g., mouse, rat, rabbit, etc) with the desiredspecificity, affinity, and capability while the constant regions arehomologous to the sequences in antibodies derived from another (usuallyhuman) to avoid eliciting an immune response in that species.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. The variable regions of the heavy andlight chain each consist of four framework regions (FR) connected bythree complementarity determining regions (CDRs) also known ashypervariable regions. The CDRs in each chain are held together in closeproximity by the FRs and, with the CDRs from the other chain, contributeto the formation of the antigen-binding site of antibodies. There are atleast two techniques for determining CDRs: (1) an approach based oncross-species sequence variability (see Kabat et al., Sequences ofProteins of Immunological Interest (5th ed., 1991, National Institutesof Health, Bethesda Md.)); and (2) an approach based on crystallographicstudies of antigen-antibody complexes (see Al-lazikani et al. J. Molec.Biol. 273:927-948 (1997)). In addition, combinations of these twoapproaches are sometimes used in the art to determine CDRs.

“Administering” is defined herein as a means of providing an agent or acomposition containing the agent to a subject in a manner that resultsin the agent being inside the subject's body. Such an administration canbe by any route including, without limitation, oral, transdermal (e.g.,vagina, rectum, oral mucosa), by injection (e.g., subcutaneous,intravenous, parenterally, intraperitoneally, intrathecal), or byinhalation (e.g., oral or nasal). Pharmaceutical preparations are, ofcourse, given by forms suitable for each administration route.

By “agent” is meant any small molecule chemical compound, antibody,nucleic acid molecule, or polypeptide, or fragments thereof.

By “analog” is meant a molecule that is not identical, but has analogousfunctional or structural features. For example, an amide, ester,carbamate, carbonate, ureide, or phosphate analog of an influenzaantibody is a molecule that either: 1) does not destroy the biologicalactivity of the influenza antibody and confers upon that influenzaantibody advantageous properties in vivo, such as uptake, duration ofaction, or onset of action; or 2) is itself biologically inactive but isconverted in vivo to a biologically active compound. Analogs includeprodrug forms of an influenza antibody. Such a prodrug is any compoundthat when administered to a biological system generates the influenzaantibody as a result of spontaneous chemical reaction(s), enzymecatalyzed chemical reaction(s), and/or metabolic chemical reaction(s).

By “control” is meant a standard or reference condition.

The term “derivative” means a pharmaceutically active compound withequivalent or near equivalent physiological functionality to a givenagent (e.g., an influenza antibody). As used herein, the term“derivative” includes any pharmaceutically acceptable salt, ether,ester, prodrug, solvate, stereoisomer including enantiomer, diastereomeror stereoisomerically enriched or racemic mixture, and any othercompound which upon administration to the recipient, is capable ofproviding (directly or indirectly) such a compound or an antivirallyactive metabolite or residue thereof.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ.

The term “epitope” or “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is a polypeptide, epitopes can be formed both from contiguousamino acids and noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained upon protein denaturing, whereas epitopes formed by tertiaryfolding are typically lost upon protein denaturing. An epitope typicallyincludes at least 3, and more usually, at least 5 or 8-10 amino acids ina unique spatial conformation.

Competition between antibodies is determined by an assay in which theimmunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen. Numerous types of competitive bindingassays are known, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology 9:242-253 (1983)); solid phase directbiotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619(1986)); solid phase direct labeled assay, solid phase direct labeledsandwich assay (see Harlow and Lane, “Antibodies, A Laboratory Manual,”Cold Spring Harbor Press (1988)); solid phase direct label RIA usingI-125 label (see Morel et al., Molec. Immunol. 25(1):7-15 (1988)); solidphase direct biotin-avidin EIA (Cheung et al., Virology 176:546-552(1990)); and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol.32:77-82 (1990)). Typically, such an assay involves the use of purifiedantigen bound to a solid surface or cells bearing either of these, anunlabeled test immunoglobulin and a labeled reference immunoglobulin.Competitive inhibition is measured by determining the amount of labelbound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excess.Antibodies identified by competition assay (competing antibodies)include antibodies binding to the same epitope as the reference antibodyand antibodies binding to an adjacent epitope sufficiently proximal tothe epitope bound by the reference antibody for steric hindrance tooccur. When a competing antibody is present in excess, it will inhibitspecific binding of a reference antibody to a common antigen by at least25%, 50, 75%, or more.

By “enhances” or “increases” is meant a positive alteration of at leastabout 10%, 25%, 50%, 75%, or 100% relative to a reference.

By “fragment” is meant a portion of a polypeptide or nucleic acidmolecule. This portion contains at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90% of the entire length of the reference nucleic acidmolecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60,70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000nucleotides or amino acids.

“Hybridization” means hydrogen bonding, which may be Watson-Crick,Hoogsteen, or reversed Hoogsteen hydrogen bonding between complementarynucleobases. For example, adenine and thymine are complementarynucleobases that pair through the formation of hydrogen bonds.

By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) thatis free of the genes which, in the naturally-occurring genome of theorganism from which the nucleic acid molecule of the invention isderived, flank the gene. The term therefore includes, for example, arecombinant DNA that is incorporated into a vector; into an autonomouslyreplicating plasmid or virus; or into the genomic DNA of a prokaryote oreukaryote; or that exists as a separate molecule (for example, a cDNA ora genomic or cDNA fragment produced by PCR or restriction endonucleasedigestion) independent of other sequences. In addition, the termincludes an RNA molecule that is transcribed from a DNA molecule, aswell as a recombinant DNA that is part of a hybrid gene encodingadditional polypeptide sequence.

By an “isolated polypeptide” is meant a polypeptide of the inventionthat has been separated from components that naturally accompany it.Typically, the polypeptide is isolated when it is at least 60%, byweight, free from the proteins and naturally-occurring organic moleculeswith which it is naturally associated. In embodiments, the preparationis at least 75%, at least 90%, or at least 99%, by weight, a polypeptideof the invention. An isolated polypeptide of the invention may beobtained, for example, by extraction from a natural source, byexpression of a recombinant nucleic acid encoding such a polypeptide; orby chemically synthesizing the protein. Purity can be measured by anyappropriate method, for example, column chromatography, polyacrylamidegel electrophoresis, HPLC analysis, and the like.

The terms “identical” or “percent identity” in the context of two ormore nucleic acids or polypeptides, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, notconsidering any conservative amino acid substitutions as part of thesequence identity. The percent identity may be measured using sequencecomparison software or algorithms or by visual inspection. Variousalgorithms and software are known in the art that may be used to obtainalignments of amino acid or nucleotide sequences. One such non-limitingexample of a sequence alignment algorithm is the algorithm described inKarlin et al., Proc. Natl. Acad. Sci., 87:2264-2268 (1990), as modifiedin Karlin et al., Proc. Natl. Acad. Sci., 90:5873-5877 (1993), andincorporated into the NBLAST and XBLAST programs (Altschul et al.,Nucleic Acids Res., 25:3389-3402 (1991)). In certain embodiments, GappedBLAST may be used as described in Altschul et al., Nucleic Acids Res.25:3389-3402 (1997). BLAST-2, WU-BLAST-2 (Altschul et al., Methods inEnzymology, 266:460-480 (1996)), ALIGN, ALIGN-2 (Genentech, South SanFrancisco, Calif.) or Megalign (DNASTAR) are additional publiclyavailable software programs that can be used to align sequences. Incertain embodiments, the percent identity between two nucleotidesequences is determined using the GAP program in GCG software (e.g.,using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90and a length weight of 1, 2, 3, 4, 5, or 6). In certain embodiments, theGAP program in the GCG software package, which incorporates thealgorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970))may be used to determine the percent identity between two amino acidsequences (e.g., using either a Blossum 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5). In certain embodiments, the percent identity betweennucleotide or amino acid sequences is determined using the algorithm ofMyers and Miller (CABIOS, 4:11-17 (1989)). For example, the percentidentity may be determined using the ALIGN program (version 2.0) andusing a PAM120 with residue table, a gap length penalty of 12 and a gappenalty of 4. Appropriate parameters for maximal alignment by particularalignment software can be determined by one skilled in the art. Incertain embodiments, the default parameters of the alignment softwareare used. In certain embodiments, the percentage identity “X” of a firstamino acid sequence to a second sequence amino acid is calculated as100×(Y/Z), where Y is the number of amino acid residues scored asidentical matches in the alignment of the first and second sequences (asaligned by visual inspection or a particular sequence alignment program)and Z is the total number of residues in the second sequence. If thelength of a first sequence is longer than the second sequence, thepercent identity of the first sequence to the second sequence will belonger than the percent identity of the second sequence to the firstsequence.

As a non-limiting example, whether any particular polynucleotide has acertain percentage sequence identity (e.g., is at least 80% identical,at least 85% identical, at least 90% identical, and in some embodiments,at least 95%, 96%, 97%, 98%, or 99% identical) to a reference sequencecan, in certain embodiments, be determined using the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2: 482 489 (1981), to find thebest segment of homology between two sequences. When using Bestfit orany other sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set such that thepercentage of identity is calculated over the full length of thereference nucleotide sequence and that gaps in homology of up to 5% ofthe total number of nucleotides in the reference sequence are allowed.

In some embodiments, two nucleic acids or polypeptides of the inventionare substantially identical, meaning they have at least 70%, at least75%, at least 80%, at least 85%, at least 90%, and in some embodimentsat least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residueidentity, when compared and aligned for maximum correspondence, asmeasured using a sequence comparison algorithm or by visual inspection.Identity can exist over a region of the sequences that is at least about5, at least about 10, about 20, about 40-60 residues in length or anyintegral value therebetween, or over a longer region than 60-80residues, at least about 90-100 residues, or the sequences aresubstantially identical over the full length of the sequences beingcompared.

A “conservative amino acid substitution” is one in which one amino acidresidue is replaced with another amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. Preferably,conservative substitutions in the sequences of the polypeptides andantibodies of the invention do not abrogate the binding of thepolypeptide or antibody containing the amino acid sequence, to theantigen(s). Methods of identifying nucleotide and amino acidconservative substitutions which do not eliminate antigen binding arewell-known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); andBurks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

“Pharmaceutically acceptable” refers to approved or approvable by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, including humans.

“Pharmaceutically acceptable excipient, carrier or diluent” refers to anexcipient, carrier or diluent that can be administered to a subject,together with an agent, and which does not destroy the pharmacologicalactivity thereof and is nontoxic when administered in doses sufficientto deliver a therapeutic amount of the agent.

A “pharmaceutically acceptable salt” of an influenza antibody recitedherein is an acid or base salt that is generally considered in the artto be suitable for use in contact with the tissues of human beings oranimals without excessive toxicity, irritation, allergic response, orother problem or complication. Such salts include mineral and organicacid salts of basic residues such as amines, as well as alkali ororganic salts of acidic residues such as carboxylic acids. Specificpharmaceutical salts include, but are not limited to, salts of acidssuch as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric,sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic,methanesulfonic, benzene sulfonic, ethane disulfonic,2-hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric,tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic,succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic,phenylacetic, alkanoic such as acetic, HOOC—(CH2)_(n)—COOH where n is0-4, and the like. Similarly, pharmaceutically acceptable cationsinclude, but are not limited to sodium, potassium, calcium, aluminum,lithium and ammonium. Those of ordinary skill in the art will recognizefurther pharmaceutically acceptable salts for the antibodies providedherein, including those listed by Remington's Pharmaceutical Sciences,17th ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985). Ingeneral, a pharmaceutically acceptable acid or base salt can besynthesized from a parent compound that contains a basic or acidicmoiety by any conventional chemical method. Briefly, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in anappropriate solvent.

The term “patient” or “subject” refers to an animal which is the objectof treatment, observation, or experiment. By way of example only, asubject includes, but is not limited to, a mammal, including, but notlimited to, a human or a non-human mammal, such as a non-human primate,bovine, equine, canine, ovine, or feline.

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment,” and the like, refer to reducing theprobability of developing a disease or condition in a subject, who doesnot have, but is at risk of or susceptible to developing a disease orcondition.

By “reduces” is meant a negative alteration of at least about 10%, 25%,50%, 75%, or 100% relative to a reference.

By “reference” is meant a standard or control condition.

By “specifically binds” is meant a compound or antibody that recognizesand binds a polypeptide of the invention, but which does notsubstantially recognize and bind other molecules in a sample, forexample, a biological sample, which naturally includes a polypeptide ofthe invention.

That an antibody “specifically binds” to an epitope or protein meansthat the antibody reacts or associates more frequently, more rapidly,with greater duration, with greater affinity, or with some combinationof the above to an epitope or protein than with alternative substances,including unrelated proteins. In certain embodiments, “specificallybinds” means, for instance, that an antibody binds to a protein with aK_(D) of about 0.1 mM or less, but more usually less than about 1 μM. Incertain embodiments, “specifically binds” means that an antibody bindsto a protein at times with a K_(D) of at least about 0.1 μM or less, andat other times at least about 0.01 μM or less. Because of the sequenceidentity between homologous proteins in different species, specificbinding can include an antibody that recognizes a particular protein inmore than one species. It is understood that an antibody or bindingmoiety that specifically binds to a first target may or may notspecifically bind to a second target. As such, “specific binding” doesnot necessarily require (although it can include) exclusive binding,i.e. binding to a single target. Generally, but not necessarily,reference to binding means specific binding.

As used herein, “substantially pure” refers to material which is atleast 50% pure (i.e., free from contaminants), more preferably at least90% pure, more preferably at least 95% pure, more preferably at least98% pure, more preferably at least 99% pure.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. By “ameliorate” is meant decrease, suppress, attenuate,diminish, arrest, or stabilize the development or progression of adisease. It will be appreciated that, although not precluded, treating adisorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

The term “therapeutic effect” refers to some extent of relief of one ormore of the symptoms of a disorder or its associated pathology. The termrefers to both therapeutic treatment and prophylactic or preventativemeasures, wherein the object is to prevent or slow down (lessen) thetargeted pathologic condition or disorder. Those in need of treatmentinclude those already with the disorder as well as those prone to havethe disorder or those in whom the disorder is to be prevented. A subjector mammal is successfully “treated” for an infection if, after receivinga therapeutic amount of an antibody according to the methods of thepresent invention, the patient shows observable and/or measurablereduction in or absence of one or more of the following: reduction inthe number of infected cells or absence of the infected cells; reductionin the percent of total cells that are infected; relief to some extentof one or more of the symptoms associated with the specific infection(e.g., symptoms associated with influenza infection); reduced morbidityand mortality, and improvement in quality of life issues. The aboveparameters for assessing successful treatment and improvement in thedisease are readily measurable by routine procedures familiar to aphysician.

“Therapeutically effective amount” is intended to qualify the amountrequired to achieve a therapeutic effect. A physician or veterinarianhaving ordinary skill in the art can readily determine and prescribe the“therapeutically effective amount” (e.g., ED₅₀) of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in apharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

The phrase “combination therapy” embraces the administration of aninfluenza antibody and a second therapeutic agent as part of a specifictreatment regimen intended to provide a beneficial effect from theco-action of these therapeutic agents. The beneficial effect of thecombination includes, but is not limited to, pharmacokinetic orpharmacodynamic co-action resulting from the combination of therapeuticagents. Administration of these therapeutic agents in combinationtypically is carried out over a defined time period (usually minutes,hours, days, or weeks depending upon the combination selected).“Combination therapy” generally is not intended to encompass theadministration of two or more of these therapeutic agents as part ofseparate monotherapy regimens that incidentally and arbitrarily resultin the combinations of the present invention. “Combination therapy” isintended to embrace administration of these therapeutic agents in asequential manner, that is, wherein each therapeutic agent isadministered at a different time, as well as administration of thesetherapeutic agents, or at least two of the therapeutic agents, in asubstantially simultaneous manner. Substantially simultaneousadministration can be accomplished, for example, by administering to thesubject a single capsule having a fixed ratio of each therapeutic agentor in multiple, single capsules for each of the therapeutic agents. Forexample, one combination of the present invention comprises an influenzaantibody and at least one additional therapeutic agent (e.g., antiviralagent, including anti-influenza agents) at the same or different timesor they can be formulated as a single, co-formulated pharmaceuticalcomposition comprising the two compounds. As another example, acombination of the present invention (e.g., an influenza antibody and atleast one additional therapeutic agent, such as an antiviral agent) isformulated as separate pharmaceutical compositions that can beadministered at the same or different time. Sequential or substantiallysimultaneous administration of each therapeutic agent can be effected byany appropriate route including, but not limited to, oral routes,intravenous routes, intramuscular routes, and direct absorption throughmucous membrane tissues (e.g., nasal, mouth, vaginal, and rectal). Thetherapeutic agents can be administered by the same route or by differentroutes. For example, one component of a particular combination may beadministered by intravenous injection while the other component(s) ofthe combination may be administered orally. The components may beadministered in any therapeutically effective sequence.

The phrase “combination” embraces groups of compounds or non-drugtherapies useful as part of a combination therapy.

The term “vector” means a construct that is capable of delivering andexpressing, one or more gene(s) or sequence(s) of interest in a hostcell. Examples of vectors include, but are not limited to, viralvectors, naked DNA or RNA expression vectors, plasmid, cosmid or phagevectors, DNA or RNA expression vectors associated with cationiccondensing agents, DNA or RNA expression vectors encapsulated inliposomes, and certain eukaryotic cells, such as producer cells.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A: Inferred lineage of clone 860. Left: the unmutated commonancestor (UCA) of the three antibodies (shown by their numbers, right)isolated from the donor. (FIG. 1B) Alignment of heavy-chain (top) (SEQID NOS 51 and 10-12, respectively, in order of appearance) andlight-chain (bottom) (SEQ ID NOS 13-16, respectively, in order ofappearance) sequences in the lineage. (FIG. 1C) Contact of the Fab fromCH65 with HA1. Heavy chain in dark blue; light chain in light blue; CDRsin colors as labeled in (FIG. 1B); HA in red, with the atomic surfaceshown as a partly transparent overlay. Residues that have mutated fromthe UCA are in green stick representation.

FIG. 2A: HA trimer with bound CH65 Fab. One HA chain is in red (HA1) andgreen (HA2); the other two chains are in gray; glycans are in yellow.The Fab bound to the colored HA chain is in dark blue (heavy chain) andlight blue (light chain), with the contacting CDRs in colors as labeledin FIG. 1B. (FIG. 2B) Blow up of the Fv region and its contact with HA1.Colors as in FIGS. 1A-1C. Note that the heavy-chain CDR3 (magenta)projects into the receptor-binding pocket on HA1, while the remainingCDRs have more limited surface contacts. (FIG. 2C) and (FIG. 2D) Surfacerepresentation of the contact between Fab CH65 and HA1, opened up asshown by the arrows. The sialic-acid pocket on one HA subunit is in darkred; the rest of the subunit, in dull red; the remaining two subunits,in gray; glycans, in yellow.

FIGS. 3A and 3B: Comparison of interactions from CH65 (FIG. 3A) andα-2,6-sialyl lactose (FIG. 3B).

FIGS. 4A-4F: Enzyme-linked immunoabsorbent assay (ELISA) of reactivityof CH65-CH67 lineage members to H1 and H3 influenza strains. 293 T cellswere transfected with full-length HA from strain X31 (H3) (top panel,FIGS. 4A-4C) or with cell-surface expressed globular head from A/SolomonIslands/3/2006 [H1] (bottom panel, FIGS. 4D-4F). Cells were fixed withformaldehyde and probed with CH65 Fab (FIGS. 4B and 4E) or CH66full-length antibody (FIGS. 4C and 4F), followed by a FITC-conjugatedsecondary antibody specific for the human Fab. Cells were imaged by FITCemission (532 nm). As a control, transfected cells were probed withsecondary antibody only (FIGS. 4A and 4D).

FIG. 5: Sequences (SEQ ID NOS 52-56, respectively, in order ofappearance) at the VDJ recombination site of CH65. The key indicates theorigin of the heavy-chain coding segments (V, D, J, and n-nucleotide).

FIG. 6: Heavy chain DNA sequences of CH65-CH67 HA antibodies.

FIG. 7: Light chain DNA sequences of CH65-CH67 HA antibodies.

FIG. 8: Heavy chain amino acid sequences of CH65-CH67 HA antibodies.

FIG. 9: Light chain amino acid sequences of CH65-CH67 HA antibodies.

FIG. 10: Alignment of VH DNA sequences of CL860UCA (SEQ ID NO: 1), CH65(SEQ ID NO: 2), CH66 (SEQ ID NO: 3) and CH67 (SEQ ID NO: 4).

FIG. 11: Alignment of VL DNA sequences of CL860UCA (SEQ ID NO: 5), CH65(SEQ ID NO: 6), CH66 (SEQ ID NO: 7) and CH67 (SEQ ID NO: 8).

FIG. 12: Alignment of VH amino acid sequences of CL860UCA (SEQ ID NO:9), CH65 (SEQ ID NO: 10), CH66 (SEQ ID NO: 11) and CH67 (SEQ ID NO: 12).

FIG. 13: Alignment of VL amino acid sequences of CL860UCA (SEQ ID NO:13), CH65 (SEQ ID NO: 14), CH66 (SEQ ID NO: 15) and CH67 (SEQ ID NO:16).

FIGS. 14A-14D: Representative receptor binding domains from H1 (FIG.14A; SEQ ID NOS 17-34, respectively, in order of appearance), H2 (FIG.14B; SEQ ID NOS 35-38, respectively, in order of appearance), H3 (FIG.14C; SEQ ID NOS 39-43, respectively, in order of appearance), and H5(FIG. 14D; (SEQ ID NOS 44-45, respectively, in order of appearance)hemagglutinin. The CH65-CH67 antibody binding epitopes are underlined.

FIG. 15: Sequence alignment of representative receptor binding domainsfrom H1, H2, H3, and H5 hemagglutinin (SEQ ID NOS 57-61, respectively,in order of appearance). The amino acid residues that interact with theCH65-CH67 antibodies are underlined.

DETAILED DESCRIPTION OF THE INVENTION

The invention features novel antibodies that broadly neutralizeinfluenza antigenic variants. The invention also provides compositionsand kits containing the novel antibodies, as well as methods for usingthese novel therapeutic molecules to treat or prevent (e.g., vaccinate)influenza infection.

The receptor for influenza virus is sialic acid, attached by terminalα-2,3 or α-2,6 linkage to glycans on glycoproteins or glycolipids(reviewed in Wiley, D. C. and Skehel, J. J. Annu. Rev. Biochem.56:365-394 (1987)). Most neutralizing antibodies block cell attachment,either because their footprint overlaps the receptor-binding site orbecause they exert steric interference when bound elsewhere on the HAsurface (Knossow, M. and Skehel, J. J. Immunology 119:1-7 (2006)). Twomouse monoclonal neutralizing antibodies, for which structures of Fab:HAcomplexes have been determined, have loops that project into thesialic-acid binding pocket on HA and present an aspartic-acid side chainroughly where the sialic-acid carboxylate would be (Fleury, D. et al.,Nat. Struct. Biol. 5:119-123 (1998); and Barbey-Martin, C. et al.,Virology 294:70-74 (2002)). But both of these antibodies also haveextensive contacts with other surface regions, in which escape mutationscould occur more readily than in the receptor site.

The invention is based, at least in part, on the discovery of novelantibodies having principal contacts in the receptor pocket. One suchantibody, designated CH65, was found by isolating rearranged heavy- andlight-chain genes from sorted single plasma cells, obtained from asubject who had received the 2007 trivalent vaccine. CH65 neutralizes aremarkably broad range of H1 seasonal isolates spanning more than threedecades. Its 19-residue heavy-chain complementarity-determining region 3(CDR-H3) inserts into the receptor pocket, mimicking many of theinteractions made by sialic acid.

Both heavy- and light-chain CDRs participate in more restricted,additional contacts with the outward-facing surface of HA1. Theinferred, unmutated ancestor of CH65 differs from the affinity maturedantibody at 12 positions in the heavy-chain variable domain, and at 6 inthe light-chain variable domain. The human B-cell repertoire thusincludes the potential to generate antibodies directed primarily at thereceptor binding site. The large number of seasonal H1 virusesneutralized by antibody CH65 suggests that such responses are ordinarilytoo rare to select for resistance, or that resistance comes at too greata fitness cost—as would be the case if potential escape mutations wereto compromise receptor binding. Thus, it is surprising that theinventors have discovered that broad neutralization of influenza viruscan be achieved by antibodies with contacts that mimic those of thereceptor. Accordingly, the invention provides novel antibodies thatmimic the contact between influenza HA and the sialic acid receptor.These novel antibodies can effectively treat and/or prevent infection bydrifted strains of influenza. As such, the invention featurescompositions and kits containing the novel antibodies, as well asmethods for using these therapeutic molecules to treat and/or preventinfluenza infection. The invention also relates to combination therapiesincluding the novel antibodies.

CH65-CH67 Hemagglutinin Antibodies

The present invention provides novel anti-influenza antibodies thatspecifically bind to an epitope of an influenza hemagglutinin (HA).Binding of the antibodies to the HA reduces or inhibits influenzahemagglutinin binding to sialic acid.

As stated above, the invention features in certain embodiments, ananti-influenza antibody or antibody fragment that specifically binds toa sialic acid binding domain of a surface antigen of influenza virus.Preferably, the surface antigen of influenza virus is HA.

In embodiments, the epitope of influenza HA comprises a sialic-acidbinding domain.

In embodiments, the HA is H1 HA, H2 HA, H3 HA, or H5 HA (an HA from ahuman adapted H5 strain).

In related embodiments, the antibody or antibody fragmenr contacts oneor more residues in the the influenza HA epitope comprising residue:158;158-160; 135-136, 190-195, and 226; 222, 225, and 227; and 187 and 189where the numbering refers to the one used in structures such as thatfor A/Solomon Islands/3/2006 (Protein Data Bank accession number 3SM5).

In one embodiment, and as stated above, the antibody contacts one ormore residues in the sialic acid binding pocket of the HA epitopeselected from the group consisting of residue: 134-136, 190-195, and226.

In another embodiment, and as stated above, the antibody furthercontacts one or more residues in the HA epitope selected from the groupconsisting of residue: 158, 158-160, 135-136, 190-195 and 226, and 187and 189.

As described herein, in certain embodiments, the antibody CDR H1 regioncontacts residue 158 of the HA epitope; the CDR H2 region contactsresidues 158-160 of the HA epitope; the CDR H3 region contacts residues135-136, 190-194 and 226 of the HA epitope; the CDR L1 region contactsresidues 222, 225 and 227 of the HA epitope; or the CDR L3 regioncontacts residues 187 and 198 of the HA epitope.

In related embodiments, the influenza HA epitope comprises the aminoacids set forth in any one of SEQ ID NOs:17-44.

In related embodiments, the influenza HA epitope comprises the CH65-CH67binding residues in any one of SEQ ID NOs:17-44 (e.g., the CH65-CH67binding residues identified in FIG. 15).

In embodiments, the anti-influenza antibody or antibody fragmentcomprises a variable heavy (V_(H)) chain, and wherein the V_(H) chaincomprises an amino acid sequence set forth in SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, or SEQ ID NO: 12.

In embodiments, the anti-influenza antibody or antibody fragmentcomprises one or more heavy chain CDR regions present in a variableheavy (V_(H)) chain amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, or SEQ ID NO: 12. In related embodiments, the one or moreheavy chain CDR regions comprises a CDR3 region present in the variableheavy (V_(H)) chain amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, or SEQ ID NO: 12.

In embodiments, the anti-influenza antibody or antibody fragmentcomprises a variable light (V_(L)) chain, and wherein the V_(L) chaincomprises an amino acid sequence set forth in SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, or SEQ ID NO: 16.

In embodiments, the anti-influenza antibody or antibody fragmentcomprises one or more light chain CDR regions present in a variablelight (V_(L)) chain amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14,SEQ ID NO: 15, or SEQ ID NO: 16. In related embodiments, the one or morelight chain CDR regions comprises a CDR3 region present in the variableheavy (V_(L)) chain amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14,SEQ ID NO: 15, or SEQ ID NO: 16.

In embodiments, the anti-influenza antibody or antibody fragmentcomprises i) a variable heavy (V_(H)) chain amino comprising an aminoacid sequence set forth in SEQ ID NO: 10, and ii) a variable light(V_(L)) chain comprising an amino acid sequence set forth in SEQ ID NO:14.

In embodiments, the anti-influenza antibody or antibody fragmentcomprises a variable heavy (V_(H)) chain, wherein the CDR3 region of theV_(H) chain comprises Arg104, Ser105, Val106, Asp107, Tyr109, Tyr110,Tyr112, or a combination thereof.

In embodiments, the anti-influenza antibody is a monoclonal antibody orantibody fragment thereof.

In embodiments, the anti-influenza antibody is a humanized antibody.

In embodiments, the antibody fragment is an Fab fragment, an Fab′fragment, an Fd fragment, a Fd′ fragment, an Fv fragment, a dAbfragment, an F(ab′)2 fragment, a single chain fragment, a diabody, or alinear antibody.

In embodiments, the anti-influenza antibody or antibody fragment furthercomprises an agent conjugated to the anti-influenza antibody or antibodyfragment thereof. In related embodiments, the agent conjugated to theantibody or antibody fragment thereof is a therapeutic agent ordetectable label.

The therapeutic agent can be any therapeutic agent suitable for use withthe novel antibodies. Such agents are well known in the art and includesmall molecules, nanoparticles, polypeptides, radioisotopes, inhibitorynucleic acids, and the like. In embodiments, the therapeutic agent is anantiviral agent or a toxin. In embodiments, the therapeutic agent is ansiRNA, shRNA, or antisense nucleic acid molecule that reduces influenzavirus production.

The detectable label can be any detectable label suitable for use withthe novel antibodies. Such labels are well known in the art and includelabels that are detected by spectroscopic, photochemical, biochemical,immunochemical, physical, or chemical means. In embodiments, thedetectable label is an enzyme, a fluorescent molecule, a particle label,an electron-dense reagent, a radiolabel, a microbubble, biotin,digoxigenin, or a hapten or a protein that has been made detectable.

In any of the above embodiments, the influenza can be H1N1, H2N2, H3N2,or a human adapted H5 strain.

The antibodies of the invention can be prepared by any conventionalmeans known in the art. For example, polyclonal antibodies can beprepared by immunizing an animal (e.g., a rabbit, rat, mouse, donkey,goat, hamster, guinea pig, sheep, ungulate, cow, camel, fowl, chicken,and the like) by multiple subcutaneous or intraperitoneal injections ofthe relevant antigen (e.g., a purified peptide fragment, full-lengthrecombinant protein, fusion protein, and the like) optionally conjugatedto suitable hapten (e.g., keyhole limpet hemocyanin (KLH), serumalbumin, and the like). The antigens can be diluted in any suitablevehicle (e.g., sterile saline) and combined with an adjuvant (e.g.,Complete or Incomplete Freund's Adjuvant) to form a stable emulsion. Thepolyclonal antibody is then recovered from blood, ascites and the like,of an animal so immunized. Collected blood is clotted, and the serumdecanted, clarified by centrifugation, and assayed for antibody titer.The polyclonal antibodies can be purified from serum or ascitesaccording to standard methods in the art including affinitychromatography, ion-exchange chromatography, gel electrophoresis,dialysis, and the like.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature 256:495 (1975). Using thehybridoma method, an appropriate host animal is immunized as describedabove to elicit the production by lymphocytes of antibodies that willspecifically bind to an immunizing antigen. Lymphocytes can also beimmunized in vitro. Following immunization, the lymphocytes are isolatedand fused with a suitable myeloma cell line using, for example,polyethylene glycol, to form hybridoma cells that can then be selectedaway from unfused lymphocytes and myeloma cells. Hybridomas that producemonoclonal antibodies directed specifically against a chosen antigen asdetermined by immunoprecipitation, immunoblotting, or by an in vitrobinding assay (e.g., radioimmunoassay (RIA), enzyme-linked immunosorbentassay (ELISA), and the like) can then be propagated either in vitroculture using standard methods (see Goding, Monoclonal Antibodies:Principles and Practice, Academic Press, 1986) or in vivo as ascitestumors in an animal. The monoclonal antibodies can then be purified fromthe culture medium or ascites fluid as described for polyclonalantibodies above.

Alternatively monoclonal antibodies can also be made using recombinantDNA methods as described in U.S. Pat. No. 4,816,567. The polynucleotidesencoding a monoclonal antibody are isolated from mature B-cells orhybridoma cell, such as by RT-PCR using oligonucleotide primers thatspecifically amplify the genes encoding the heavy and light chains ofthe antibody, and their sequence is determined using conventionalprocedures. The isolated polynucleotides (including the isolatedpolynucleotides described herein) encoding the heavy and light chainsare then cloned into suitable expression vectors, which when transfectedinto host cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, monoclonal antibodies are generated by the hostcells. Also, recombinant monoclonal antibodies or fragments thereof ofthe desired species can be isolated from phage display librariesexpressing CDRs of the desired species as described (see McCafferty etal., 1990, Nature, 348:552-554; Clackson et al., 1991, Nature,352:624-628; and Marks et al., 1991, J. Mol. Biol., 222:581-597).

The polynucleotide(s) encoding a monoclonal antibody can further bemodified in a number of different manners using recombinant DNAtechnology to generate alternative antibodies. In some embodiments, theconstant domains of the light and heavy chains of, for example, a mousemonoclonal antibody can be substituted 1) for those regions of, forexample, a human antibody to generate a chimeric antibody or 2) for anon-immunoglobulin polypeptide to generate a fusion antibody. In someembodiments, the constant regions are truncated or removed to generatethe desired antibody fragment of a monoclonal antibody. Site-directed orhigh-density mutagenesis of the variable region can be used to optimizespecificity, affinity, etc. of a monoclonal antibody.

In some embodiments of the present invention, the monoclonal antibody isa humanized antibody. Humanized antibodies are antibodies that containminimal sequences from non-human (e.g., rodent) antibodies within theantigen determination or hypervariable region that comprise the threecomplementary determination regions (CDRs) within each antibody chain.Such antibodies are used therapeutically to reduce antigenicity and HAMA(human anti-mouse antibody) responses when administered to a humansubject. In practice, humanized antibodies are typically humanantibodies with minimum to virtually no non-human sequences. A humanantibody is an antibody produced by a human or an antibody having anamino acid sequence corresponding to an antibody produced by a human.

Humanized antibodies can be produced using various techniques known inthe art. An antibody can be humanized by substituting the CDRs of ahuman antibody with those of a non-human antibody (e.g., mouse, rat,rabbit, hamster, and the like) having the desired specificity, affinity,and capability (see, e.g., the methods of Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); andVerhoeyen et al., Science 239:1534-1536 (1988). The humanized antibodycan be further modified by the substitution of additional residue eitherin the variable human framework region and/or within the replacednon-human residues to refine and optimize antibody specificity,affinity, and/or capability.

The choice of human heavy and/or light chain variable domains to be usedin making humanized antibodies can be important for reducingantigenicity. According to the “best-fit” method, the sequence of thevariable domain of a non-human antibody is screened against the entirelibrary of known human variable-domain amino acid sequences. Thus incertain embodiments, the human amino acid sequence which is mosthomologous to that of the non-human antibody from which the CDRs aretaken is used as the human framework region (FR) for the humanizedantibody (see Sims et al., J. Immunol. 151: 2296 (1993); Chothia et al.,J. Mol. Biol. 196:901 (1987)). Another method uses a particular FRderived from the consensus sequence of all human antibodies of aparticular subgroup of light or heavy chains and can be used for severaldifference humanized antibodies (see Carter et al., PNAS 89; 4285(1992); Presta et al., J. Immunol. 151: 2623 (1993)). In embodiments, acombination of methods is used to pick the human variable FR to use ingeneration of humanized antibodies.

It is further understood that antibodies to be humanized must retainhigh affinity for the antigen as well as other favorable biologicalproperties. To achieve this goal, humanized antibodies can be preparedby a process of analysis of the parental sequence from the non-humanantibody to be humanized and the various candidate humanizing sequences.Three-dimensional immunoglobulin models are available and familiar tothose skilled in the art. Computer programs can be used to illustrateand display probable three-dimensional conformational structures ofselected candidate antibody sequences. Use of such models permitsanalysis of the likely role of the residues in the function of theantibody to be humanized, i.e., the analysis of residues that influencethe ability of the candidate antibody to bind its antigen. In this way,FR residues can be selected and combined from the parental antibody tothe recipient humanized antibody so that the desired antibodycharacteristics are achieved. In general, the residues in the CDRs ofthe antigen determination region (or hypervariable region) are retainedfrom the parental antibody (e.g. the non-human antibody with the desiredantigen binding properties) in the humanized antibody for antigenbinding. In certain embodiments, at least one additional residue withinthe variable FR is retained from the parental antibody in the humanizedantibody. In certain embodiments, up to six additional residues withinthe variable FR are retained from the parental antibody in the humanizedantibody.

Amino acids from the variable regions of the mature heavy and lightchains of immunoglobulins are designated Hx and Lx respectively, where xis a number designating the position of an amino acid according to thescheme of Kabat, Sequences of Proteins of Immunological Interest, U.S.Department of Health and Human Services, 1987, 1991. Kabat lists manyamino acid sequences for antibodies for each subgroup, and lists themost commonly occurring amino acid for each residue position in thatsubgroup to generate a consensus sequence. Kabat uses a method forassigning a residue number to each amino acid in a listed sequence, andthis method for assigning residue numbers has become standard in thefield. Kabat's scheme is extendible to other antibodies not included inhis compendium by aligning the antibody in question with one of theconsensus sequences in Kabat by reference to conserved amino acids. Theuse of the Kabat numbering system readily identifies amino acids atequivalent positions in different antibodies. For example, an amino acidat the L50 position of a human antibody occupies the equivalent positionto an amino acid position L50 of a mouse antibody. Moreover, any twoantibody sequences can be uniquely aligned, for example to determinepercent identity, by using the Kabat numbering system so that each aminoacid in one antibody sequence is aligned with the amino acid in theother sequence that has the same Kabat number. After alignment, if asubject antibody region (e.g., the entire mature variable region of aheavy or light chain) is being compared with the same region of areference antibody, the percentage sequence identity between the subjectand reference antibody regions is the number of positions occupied bythe same amino acid in both the subject and reference antibody regiondivided by the total number of aligned positions of the two regions,with gaps not counted, multiplied by 100 to convert to percentage.

In addition to humanized antibodies, fully human antibodies can bedirectly prepared using various techniques known in the art.Immortalized human B lymphocytes immunized in vitro or isolated from animmunized individual that produce an antibody directed against a targetantigen can be generated (See, e.g., Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., J.Immunol. 147:86-95 (1991); and U.S. Pat. No. 5,750,373). Also, the humanantibody can be selected from a phage library, where that phage libraryexpresses human antibodies (see Vaughan et al., Nat. Biotech. 14:309-314(1996); Sheets et al., Proc. Nat'l. Acad. Sci. 95:6157-6162 (1998);Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); and Marks et al.,J. Mol. Biol. 222:581 (1991)). Human antibodies can also be made intransgenic mice containing human immunoglobulin loci that are capableupon immunization of producing the full repertoire of human antibodiesin the absence of endogenous immunoglobulin production. This approach isdescribed in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; and 5,661,016.

This invention also encompasses bispecific antibodies. Bispecificantibodies are antibodies that are capable of specifically recognizingand binding at least two different epitopes. The different epitopes caneither be within the same molecule (e.g., influenza HA) or on differentmolecules such that the bispecific antibody can specifically recognizeand bind an epitope in an antigen of interest (e.g., influenza HA) aswell as, for example, another viral protein (e.g., neurominidase, M2,and the like). Bispecific antibodies can be intact antibodies orantibody fragments. Techniques for making bispecific antibodies arecommon in the art (see Millstein et al., Nature 305:537-539 (1983);Brennan et al., Science 229:81 (1985); Suresh et al, Methods in Enzymol.121:120 (1986); Traunecker et al., EMBO J. 10:3655-3659 (1991); Shalabyet al., J. Exp. Med. 175:217-225 (1992); Kostelny et al., J. Immunol.148:1547-1553 (1992); Gruber et al., J. Immunol. 152:5368 (1994); andU.S. Pat. No. 5,731,168). Antibodies with more than two valencies arealso contemplated. For example, trispecific antibodies can be prepared(see Tutt et al., J. Immunol. 147:60 (1991)).

In embodiments, the antibodies of the invention are antibody fragments.Various techniques are known for the production of antibody fragments:Traditionally, these fragments are derived via proteolytic digestion ofintact antibodies (see, e.g., Morimoto et al., Journal of Biochemicaland Biophysical Methods 24:107-117 (1993); and Brennan et al., Science229:81 (1985)). Antibody fragments can also be produced recombinantly.Fab, Fv, and scFv antibody fragments can all be expressed in andsecreted from E. coli or other host cells, thus allowing the productionof large amounts of these fragments. Such antibody fragments can also beisolated from antibody phage libraries as discussed above. The antibodyfragment can also be linear antibodies as described in U.S. Pat. No.5,641,870, for example, and can be monospecific or bispecific. Othertechniques for the production of antibody fragments will be readilyapparent to the skilled practitioner.

According to the present invention, techniques can be adapted for theproduction of single-chain antibodies specific to a polypeptide of theinvention (see U.S. Pat. No. 4,946,778). In addition, methods can beadapted for the construction of Fab expression libraries (see Huse etal., Science 246:1275-1281 (1989)) to allow rapid and effectiveidentification of monoclonal Fab fragments with the desired specificityfor influenza HA.

Antibody fragments that contain the idiotypes to a polypeptide of theinvention may be produced by techniques in the art including, but notlimited to: (a) an F(ab′)2 fragment produced by pepsin digestion of anantibody molecule; (b) an Fab fragment generated by reducing thedisulfide bridges of an F(ab′)2 fragment, (c) an Fab fragment generatedby the treatment of the antibody molecule with papain and a reducingagent, and (d) Fv fragments.

It can further be desirable, especially in the case of antibodyfragments, to modify an antibody in order to increase its serumhalf-life. This can be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment by mutationof the appropriate region in the antibody fragment or by incorporatingthe epitope into a peptide tag that is then fused to the antibodyfragment at either end or in the middle (e.g., by DNA or peptidesynthesis).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune cells to unwanted cells (U.S. Pat. No. 4,676,980). It iscontemplated that the antibodies can be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins can be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

Another alteration contemplated by the present invention is that thevariable domains in both the heavy and light chains are altered by atleast partial replacement of one or more CDRs and, if necessary, bypartial framework region replacement and sequence changing. Although theCDRs may be derived from an antibody of the same class or even subclassas the antibody from which the framework regions are derived, it isenvisaged that the CDRs will be derived from an antibody of differentclass and preferably from an antibody from a different species. It maynot be necessary to replace all of the CDRs with the complete CDRs fromthe donor variable region to transfer the antigen binding capacity ofone variable domain to another. Rather, it may only be necessary totransfer those residues that are necessary to maintain the activity ofthe antigen binding site. Given the explanations set forth in U.S. Pat.Nos. 5,585,089, 5,693,761 and 5,693,762, it will be well within thecompetence of those skilled in the art, either by carrying out routineexperimentation or by trial and error testing to obtain afuncpQE-9tional antibody with reduced immunogenicity.

Alterations to the variable region notwithstanding, those skilled in theart will appreciate that the modified antibodies of this invention cancomprise antibodies, or immunoreactive fragments thereof, in which atleast a fraction of one or more of the constant region domains has beendeleted or otherwise altered so as to provide desired biochemicalcharacteristics such as increased tumor localization or reduced serumhalf-life when compared with an antibody of approximately the sameimmunogenicity comprising a native or unaltered constant region. In someembodiments, the constant region of the modified antibodies willcomprise a human constant region. Modifications to the constant regioncompatible with this invention comprise additions, deletions orsubstitutions of one or more amino acids in one or more domains. Thatis, the modified antibodies disclosed herein may comprise alterations ormodifications to one or more of the three heavy chain constant domains(CH1, CH2 or CH3) and/or to the light chain constant domain (CL). Insome embodiments of the invention modified constant regions wherein oneor more domains are partially or entirely deleted are contemplated. Insome embodiments the modified antibodies will comprise domain deletedconstructs or variants wherein the entire CH2 domain has been removed(ΔCH2 constructs). In some embodiments the omitted constant regiondomain will be replaced by a short amino acid spacer (e.g., 10 residues)that provides some of the molecular flexibility typically imparted bythe absent constant region.

The present invention further embraces variants and equivalents whichare substantially homologous to the chimeric, humanized and humanantibodies, or antibody fragments thereof, set forth herein. These cancontain, for example, conservative substitution mutations, i.e., thesubstitution of one or more amino acids by similar amino acids. Forexample, conservative substitution refers to the substitution of anamino acid with another within the same general class such as, forexample, one acidic amino acid with another acidic amino acid, one basicamino acid with another basic amino acid or one neutral amino acid byanother neutral amino acid. What is intended by a conservative aminoacid substitution is well known in the art.

The antibodies of the present invention can be assayed forimmunospecific binding by any method known in the art. The immunoassayswhich can be used include, but are not limited to, competitive andnon-competitive assay systems using techniques such as BIAcore analysis,FACS analysis, immunofluorescence, immunocytochemistry, Western blots,radioimmunoassays, ELISA, “sandwich” immunoassays, immunoprecipitationassays, precipitin reactions, gel diffusion precipitin reactions,immunodiffusion assays, agglutination assays, complement-fixationassays, immunoradiometric assays, fluorescent immunoassays, protein Aimmunoassays, and the like. Such assays are routine and well known inthe art (see, e.g., Ausubel et al, eds, 1994, Current Protocols inMolecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which isincorporated by reference herein in its entirety).

In embodiments, the immunospecificity of an antibody against a influenzaHA is determined using ELISA. An ELISA assay comprises preparingantigen, coating wells of a microtiter plate with antigen, adding theantibody conjugated to a detectable compound such as an enzymaticsubstrate (e.g., horseradish peroxidase or alkaline phosphatase) to thewell, incubating for a period of time and detecting the presence of theantigen. In some embodiments, the antibody is not conjugated to adetectable compound, but instead a second conjugated antibody thatrecognizes the antibody against the influenza HA antigen is added to thewell. In some embodiments, instead of coating the well with the antigen,the antibody can be coated to the well and a second antibody conjugatedto a detectable compound can be added following the addition of theantigen to the coated well. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thesignal detected as well as other variations of ELISAs known in the art(see e.g. Ausubel et al, eds, 1994, Current Protocols in MolecularBiology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1).

The binding affinity of an antibody to influenza HA and the off-rate ofan antibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g. ³H or ¹²⁵I), orfragment or variant thereof, with the antibody of interest in thepresence of increasing amounts of unlabeled antigen followed by thedetection of the antibody bound to the labeled antigen. The affinity ofthe antibody against an antigen and the binding off-rates can bedetermined from the data by scatchard plot analysis. In someembodiments, BIAcore kinetic analysis is used to determine the bindingon and off rates of antibodies against a cancer stem cell marker.BIAcore kinetic analysis comprises analyzing the binding anddissociation of antibodies from chips with immobilized cancer stem cellmarker antigens on their surface.

Influenza Hemagglutinin Antibody Polypeptides and Polynucleotides

The present invention also encompasses isolated polynucleotides thatencode a polypeptide comprising an influenza hemagglutinin (HA) antibodyor fragment thereof.

The term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequences for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequences. The polynucleotides of the invention can be in theform of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, andsynthetic DNA; and can be double-stranded or single-stranded, and ifsingle stranded can be the coding strand or non-coding (anti-sense)strand.

The present invention further relates to variants of thepolynucleotides, for example, fragments, analogs, and derivatives. Thevariant of the polynucleotide can be a naturally occurring allelicvariant of the polynucleotide or a non-naturally occurring variant ofthe polynucleotide. In certain embodiments, the polynucleotide can havea coding sequence which is a naturally occurring allelic variant of thecoding sequence of the disclosed polypeptides. As known in the art, anallelic variant is an alternate form of a polynucleotide sequence thathave, for example, a substitution, deletion, or addition of one or morenucleotides, which does not substantially alter the function of theencoded polypeptide.

In embodiments, the polynucleotides can comprise the coding sequence forthe mature polypeptide fused in the same reading frame to apolynucleotide which aids, for example, in expression and secretion of apolypeptide from a host cell (e.g., a leader sequence which functions asa secretory sequence for controlling transport of a polypeptide from thecell). The polypeptide having a leader sequence is a preprotein and canhave the leader sequence cleaved by the host cell to form the matureform of the polypeptide. The polynucleotides can also encode for aproprotein which is the mature protein plus additional 5′ amino acidresidues. A mature protein having a prosequence is a proprotein and isan inactive form of the protein. Once the prosequence is cleaved anactive mature protein remains.

In embodiments, the polynucleotides can comprise the coding sequence forthe mature polypeptide fused in the same reading frame to a markersequence that allows, for example, for purification of the encodedpolypeptide. For example, the marker sequence can be a hexa-histidinetag (SEQ ID NO: 46) supplied by a pQE-9 vector to provide forpurification of the mature polypeptide fused to the marker in the caseof a bacterial host, or the marker sequence can be a hemagglutinin (HA)tag derived from the influenza hemagglutinin protein when a mammalianhost (e.g., COS-7 cells) is used. Additional tags include, but are notlimited to, Calmodulin tags, FLAG tags, Myc tags, S tags, SBP tags,Softag 1, Softag 3, V5 tag, Xpress tag, Isopeptag, SpyTag, BiotinCarboxyl Carrier Protein (BCCP) tags, GST tags, fluorescent protein tags(e.g., green fluorescent protein tags), maltose binding protein tags,Nus tags, Strep-tag, thioredoxin tag, TC tag, Ty tag, and the like.

In embodiments, the present invention provides isolated nucleic acidmolecules having a nucleotide sequence at least 80% identical, at least85% identical, at least 90% identical, at least 95% identical, or atleast 96%, 97%, 98% or 99% identical to a polynucleotide encoding apolypeptide comprising an influenza HA antibody or antibody fragment ofthe present invention.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence can include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto 5% of the nucleotides in the reference sequence can be deleted orsubstituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence can be inserted intothe reference sequence. These mutations of the reference sequence canoccur at the amino- or carboxy-terminal positions of the referencenucleotide sequence or anywhere between those terminal positions,interspersed either individually among nucleotides in the referencesequence or in one or more contiguous groups within the referencesequence.

As a practical matter, whether any particular nucleic acid molecule isat least 80% identical, at least 85% identical, at least 90% identical,and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% identicalto a reference sequence can be determined conventionally using knowncomputer programs such as the Bestfit program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).Bestfit uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2: 482 489 (1981), to find the bestsegment of homology between two sequences. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set such that thepercentage of identity is calculated over the full length of thereference nucleotide sequence and that gaps in homology of up to 5% ofthe total number of nucleotides in the reference sequence are allowed.

The polynucleotide variants can contain alterations in the codingregions, non-coding regions, or both. In some embodiments, thepolynucleotide variants contain alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. In some embodiments,nucleotide variants are produced by silent substitutions due to thedegeneracy of the genetic code. Polynucleotide variants can be producedfor a variety of reasons, e.g., to optimize codon expression for aparticular host (change codons in the human mRNA to those preferred by abacterial host such as E. coli).

The polypeptides of the present invention can be recombinantpolypeptides, natural polypeptides, or synthetic polypeptides comprisingan antibody, or fragment thereof, against influenza HA. It will berecognized in the art that some amino acid sequences of the inventioncan be varied without significant effect of the structure or function ofthe protein. Thus, the invention further includes variations of thepolypeptides which show substantial activity or which include regions ofa humanized antibody, or fragment thereof, against influenza HA. Suchmutants include deletions, insertions, inversions, repeats, and typesubstitutions.

The polypeptides and analogs can be further modified to containadditional chemical moieties not normally part of the protein. Thosederivatized moieties can improve the solubility, the biological halflife or absorption of the protein. The moieties can also reduce oreliminate any desirable side effects of the proteins and the like. Anoverview for those moieties can be found in Remington's PharmaceuticalSciences, 20th ed., Mack Publishing Co., Easton, Pa. (2000).

The isolated polypeptides described herein can be produced by anysuitable method known in the art. Such methods range from direct proteinsynthetic methods to constructing a DNA sequence encoding isolatedpolypeptide sequences and expressing those sequences in a suitabletransformed host. In some embodiments, a DNA sequence is constructedusing recombinant technology by isolating or synthesizing a DNA sequenceencoding a wild-type protein of interest. Optionally, the sequence canbe mutagenized by site-specific mutagenesis to provide functionalanalogs thereof. See, e.g. Zoeller et al., Proc. Nat'l. Acad. Sci. USA81:5662-5066 (1984) and U.S. Pat. No. 4,588,585.

In embodiments, a DNA sequence encoding a polypeptide of interest wouldbe constructed by chemical synthesis using an oligonucleotidesynthesizer. Such oligonucleotides can be designed based on the aminoacid sequence of the desired polypeptide and selecting those codons thatare favored in the host cell in which the recombinant polypeptide ofinterest will be produced. Standard methods can be applied to synthesizean isolated polynucleotide sequence encoding an isolated polypeptide ofinterest. For example, a complete amino acid sequence can be used toconstruct a back-translated gene. Further, a DNA oligomer containing anucleotide sequence coding for the particular isolated polypeptide canbe synthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.The individual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly.

Once assembled (e.g., by synthesis, site-directed mutagenesis, oranother method), the polynucleotide sequences encoding a particularisolated polypeptide of interest will be inserted into an expressionvector and optionally operatively linked to an expression controlsequence appropriate for expression of the protein in a desired host.Proper assembly can be confirmed by nucleotide sequencing, restrictionmapping, and expression of a biologically active polypeptide in asuitable host. As well known in the art, in order to obtain highexpression levels of a transfected gene in a host, the gene can beoperatively linked to transcriptional and translational expressioncontrol sequences that are functional in the chosen expression host.

Recombinant expression vectors are used to amplify and express DNAencoding the influenza HA antibodies. Recombinant expression vectors arereplicable DNA constructs which have synthetic or cDNA-derived DNAfragments encoding an influenza HA antibody or a bioequivalent analogoperatively linked to suitable transcriptional or translationalregulatory elements derived from mammalian, microbial, viral or insectgenes. A transcriptional unit generally comprises an assembly of (1) agenetic element or elements having a regulatory role in gene expression,for example, transcriptional promoters or enhancers, (2) a structural orcoding sequence which is transcribed into mRNA and translated intoprotein, and (3) appropriate transcription and translation initiationand termination sequences, as described in detail below. Such regulatoryelements can include an operator sequence to control transcription. Theability to replicate in a host, usually conferred by an origin ofreplication, and a selection gene to facilitate recognition oftransformants can additionally be incorporated. DNA regions areoperatively linked when they are functionally related to each other. Forexample, DNA for a signal peptide (secretory leader) is operativelylinked to DNA for a polypeptide if it is expressed as a precursor whichparticipates in the secretion of the polypeptide; a promoter isoperatively linked to a coding sequence if it controls the transcriptionof the sequence; or a ribosome binding site is operatively linked to acoding sequence if it is positioned so as to permit translation.Generally, operatively linked means contiguous, and in the case ofsecretory leaders, means contiguous and in reading frame. Structuralelements intended for use in yeast expression systems include a leadersequence enabling extracellular secretion of translated protein by ahost cell. Alternatively, where recombinant protein is expressed withouta leader or transport sequence, it can include an N-terminal methionineresidue. This residue can optionally be subsequently cleaved from theexpressed recombinant protein to provide a final product.

The choice of expression control sequence and expression vector willdepend upon the choice of host. A wide variety of expression host/vectorcombinations can be employed. Useful expression vectors for eukaryotichosts, include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovirus andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from Escherichia coli,including pCR 1, pBR322, pMB9 and their derivatives, wider host rangeplasmids, such as M13 and filamentous single-stranded DNA phages.

Suitable host cells for expression of a polypeptide include prokaryotes,yeast, insect or higher eukaryotic cells under the control ofappropriate promoters. Prokaryotes include gram negative or grampositive organisms, for example E. coli or bacilli. Higher eukaryoticcells include established cell lines of mammalian origin. Cell-freetranslation systems could also be employed. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are well known in the art (see Pouwels et al., CloningVectors: A Laboratory Manual, Elsevier, N.Y., 1985).

Various mammalian or insect cell culture systems are also advantageouslyemployed to express recombinant protein. Expression of recombinantproteins in mammalian cells can be performed because such proteins aregenerally correctly folded, appropriately modified and completelyfunctional. Examples of suitable mammalian host cell lines include theCOS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175,1981), and other cell lines capable of expressing an appropriate vectorincluding, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO),HeLa and BHK cell lines. Mammalian expression vectors can comprisenontranscribed elements such as an origin of replication, a suitablepromoter and enhancer linked to the gene to be expressed, and other 5′or 3′ flanking nontranscribed sequences, and 5′ or 3′ nontranslatedsequences, such as necessary ribosome binding sites, a polyadenylationsite, splice donor and acceptor sites, and transcriptional terminationsequences. Baculovirus systems for production of heterologous proteinsin insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47(1988).

The proteins produced by a transformed host can be purified according toany suitable method. Such standard methods include chromatography (e.g.,ion exchange, affinity and sizing column chromatography, and the like),centrifugation, differential solubility, or by any other standardtechnique for protein purification. Affinity tags such as hexahistidine,maltose binding domain, influenza coat sequence,glutathione-S-transferase, and the like can be attached to the proteinto allow easy purification by passage over an appropriate affinitycolumn. Isolated proteins can also be physically characterized usingsuch techniques as proteolysis, nuclear magnetic resonance and x-raycrystallography.

For example, supernatants from systems which secrete recombinant proteininto culture media can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a suitable purification matrix.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a cancer stem cell protein-Fc composition.Some or all of the foregoing purification steps, in variouscombinations, can also be employed to provide a homogeneous recombinantprotein.

Recombinant protein produced in bacterial culture can be isolated, forexample, by initial extraction from cell pellets, followed by one ormore concentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. High performance liquid chromatography (HPLC) canbe employed for final purification steps. Microbial cells employed inexpression of a recombinant protein can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Methods of Treatment

The present invention provides methods for treating or preventinginfluenza infection.

In aspects, the invention provides methods for treating or preventing aninfluenza virus infection in a subject in need thereof. The methodsinvolve administering to the subject an effective amount of ananti-influenza antibody or antibody fragment described herein, or apharmaceutical composition containing the antibody or antibody fragment.The methods treat or prevent influenza virus infection in the subject,including reducing or alleviating symptoms associated with infection.

In aspects, the invention provides methods for neutralizing an influenzavirus in a subject in need thereof. The methods involve administering tothe subject an effective amount of an anti-influenza antibody orantibody fragment described herein, or a pharmaceutical compositioncontaining the antibody or antibody fragment. The methods neutralize theinfluenza virus in the subject, thereby treating or prevent influenzavirus infection in the subject, including reducing or alleviatingsymptoms associated with infection.

In aspects, the invention provides methods for establishment ofinfluenza virus infection in a subject in need thereof. The methodsinvolve administering to the subject an effective amount of ananti-influenza antibody or antibody fragment described herein, or apharmaceutical composition containing the antibody or antibody fragment.The methods inhibit establishment of influenza virus infection in thesubject, thereby preventing symptoms associated with infection.

In aspects, the invention provides methods for inhibiting disseminationof influenza virus infection in a subject in need thereof. The methodsinvolve administering to the subject an effective amount of ananti-influenza antibody or antibody fragment described herein, or apharmaceutical composition containing the antibody or antibody fragment.The methods inhibit dissemination of influenza virus infection in thesubject, thereby reducing or alleviating symptoms associated withinfection.

In aspects, the invention provides methods for inhibiting influenzavirus entry into a cell in a subject. The methods involve administeringto the subject an effective amount of an anti-influenza antibody orantibody fragment described herein, or a pharmaceutical compositioncontaining the antibody or antibody fragment. The methods inhibitinfluenza virus entry into a cell in the subject, thereby preventingsymptoms associated with infection or reducing or alleviating symptomsassociated with infection.

In aspects, the invention provides methods for inhibiting influenzavirus entry into a cell. The methods involve contacting a cell having orat risk of developing influenza virus infection with an anti-influenzaantibody or antibody fragment described herein, or a pharmaceuticalcomposition containing the antibody or antibody fragment. The methodsinhibit influenza virus entry into the cell.

In any of the above aspects, the influenza can be H1N1, H2N2, H3N2, or ahuman adapted H5 strain.

In any of the above aspects, the subject has or is at risk of developingan influenza infection. In related embodiments, the subject is a mammal(e.g., human). In related embodiments, the subject is susceptible toviral infection (e.g., a pregnant female, a young subject or an infantsubject, an elderly subject).

In any of the above aspects and embodiments, the anti-influenza antibodyor antibody fragment, or the pharmaceutical composition is administeredby intramuscular injection, intravenous injection, subcutaneousinjection, or inhalation.

Pharmaceutical Compositions

The invention provides for pharmaceutical compositions containing thenovel influenza HA antibodies described herein. In embodiments, thepharmaceutical compositions contain a pharmaceutically acceptablecarrier, excipient, or diluent, which includes any pharmaceutical agentthat does not itself induce the production of an immune response harmfulto a subject receiving the composition, and which may be administeredwithout undue toxicity. As used herein, the term “pharmaceuticallyacceptable” means being approved by a regulatory agency of the Federalor a state government or listed in the U.S. Pharmacopia, EuropeanPharmacopia or other generally recognized pharmacopia for use inmammals, and more particularly in humans. These compositions can beuseful for treating and/or preventing influenza infection.

A thorough discussion of pharmaceutically acceptable carriers, diluents,and other excipients is presented in Remington's Pharmaceutical Sciences(17th ed., Mack Publishing Company) and Remington: The Science andPractice of Pharmacy (21st ed., Lippincott Williams & Wilkins), whichare hereby incorporated by reference. The formulation of thepharmaceutical composition should suit the mode of administration. Inembodiments, the pharmaceutical composition is suitable foradministration to humans, and can be sterile, non-particulate and/ornon-pyrogenic.

Pharmaceutically acceptable carriers, excipients, or diluents include,but are not limited, to saline, buffered saline, dextrose, water,glycerol, ethanol, sterile isotonic aqueous buffer, and combinationsthereof.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives, and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include, but arenot limited to: (1) water soluble antioxidants, such as ascorbic acid,cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodiumsulfite and the like; (2) oil-soluble antioxidants, such as ascorbylpalmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene(BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3)metal chelating agents, such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

In embodiments, the pharmaceutical composition is provided in a solidform, such as a lyophilized powder suitable for reconstitution, a liquidsolution, suspension, emulsion, tablet, pill, capsule, sustained releaseformulation, or powder.

In embodiments, the pharmaceutical composition is supplied in liquidform, for example, in a sealed container indicating the quantity andconcentration of the active ingredient in the pharmaceuticalcomposition. In related embodiments, the liquid form of thepharmaceutical composition is supplied in a hermetically sealedcontainer.

Methods for formulating the pharmaceutical compositions of the presentinvention are conventional and well-known in the art (see Remington andRemington's). One of skill in the art can readily formulate apharmaceutical composition having the desired characteristics (e.g.,route of administration, biosafety, and release profile).

Methods for preparing the pharmaceutical compositions include the stepof bringing into association the active ingredient with apharmaceutically acceptable carrier and, optionally, one or moreaccessory ingredients. The pharmaceutical compositions can be preparedby uniformly and intimately bringing into association the activeingredient with liquid carriers, or finely divided solid carriers, orboth, and then, if necessary, shaping the product. Additionalmethodology for preparing the pharmaceutical compositions, including thepreparation of multilayer dosage forms, are described in Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems (9th ed.,Lippincott Williams & Wilkins), which is hereby incorporated byreference.

Methods of Delivery

The pharmaceutical compositions of the invention can be administered toa subject by oral and non-oral means (e.g., topically, transdermally, orby injection). Such modes of administration and the methods forpreparing an appropriate pharmaceutical composition for use therein aredescribed in Gibaldi's Drug Delivery Systems in Pharmaceutical Care (1sted., American Society of Health-System Pharmacists), which is herebyincorporated by reference.

In embodiments, the pharmaceutical compositions are administered orallyin a solid form.

Pharmaceutical compositions suitable for oral administration can be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound(s) describedherein, a derivative thereof, or a pharmaceutically acceptable salt orprodrug thereof as the active ingredient(s). The active ingredient canalso be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (e.g., capsules, tablets,pills, dragees, powders, granules and the like), the active ingredientis mixed with one or more pharmaceutically acceptable carriers,excipients, or diluents, such as sodium citrate or dicalcium phosphate,and/or any of the following: (1) fillers or extenders, such as starches,lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders,such as, for example, carboxymethylcellulose, alginates, gelatin,polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such asglycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate; (5) solution retarding agents, such as paraffin;(6) absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, acetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets, and pills, thepharmaceutical compositions can also comprise buffering agents. Solidcompositions of a similar type can also be prepared using fillers insoft and hard-filled gelatin capsules, and excipients such as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet can be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets can be prepared usingbinders (for example, gelatin or hydroxypropylmethyl cellulose),lubricants, inert diluents, preservatives, disintegrants (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-actives, and/or dispersing agents. Molded tablets can be made bymolding in a suitable machine a mixture of the powdered activeingredient moistened with an inert liquid diluent.

The tablets and other solid dosage forms, such as dragees, capsules,pills, and granules, can optionally be scored or prepared with coatingsand shells, such as enteric coatings and other coatings well-known inthe art.

The pharmaceutical compositions can also be formulated so as to provideslow, extended, or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. The pharmaceutical compositions can alsooptionally contain opacifying agents and may be of a composition thatreleases the active ingredient(s) only, or preferentially, in a certainportion of the gastrointestinal tract, optionally, in a delayed manner.Examples of embedding compositions include polymeric substances andwaxes. The active ingredient can also be in micro-encapsulated form, ifappropriate, with one or more pharmaceutically acceptable carriers,excipients, or diluents well-known in the art (see, e.g., Remington andRemington's).

The pharmaceutical compositions can be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use.

In embodiments, the pharmaceutical compositions are administered orallyin a liquid form.

Liquid dosage forms for oral administration of an active ingredientinclude pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms can contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof. In addition to inert diluents,the liquid pharmaceutical compositions can include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents, and the like.

Suspensions, in addition to the active ingredient(s) can containsuspending agents such as, but not limited to, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

In embodiments, the pharmaceutical compositions are administered bynon-oral means such as by topical application, transdermal application,injection, and the like. In related embodiments, the pharmaceuticalcompositions are administered parenterally by injection, infusion, orimplantation (e.g., intravenous, intramuscular, intraarticular,subcutaneous, and the like).

Compositions for parenteral use can be presented in unit dosage forms,e.g. in ampoules or in vials containing several doses, and in which asuitable preservative can be added. Such compositions can be in form ofa solution, a suspension, an emulsion, an infusion device, a deliverydevice for implantation, or it can be presented as a dry powder to bereconstituted with water or another suitable vehicle before use. One ormore co-vehicles, such as ethanol, can also be employed. Apart from theactive ingredient(s), the compositions can contain suitable parenterallyacceptable carriers and/or excipients or the active ingredient(s) can beincorporated into microspheres, microcapsules, nanoparticles, liposomes,or the like for controlled release. Furthermore, the compositions canalso contain suspending, solubilising, stabilising, pH-adjusting agents,and/or dispersing agents.

The pharmaceutical compositions can be in the form of sterileinjections. To prepare such a composition, the active ingredient isdissolved or suspended in a parenterally acceptable liquid vehicle.Exemplary vehicles and solvents include, but are not limited to, water,water adjusted to a suitable pH by addition of an appropriate amount ofhydrochloric acid, sodium hydroxide or a suitable buffer,1,3-butanediol, Ringer's solution and isotonic sodium chloride solution.The pharmaceutical composition can also contain one or morepreservatives, for example, methyl, ethyl or n-propyl p-hydroxybenzoate.To improve solubility, a dissolution enhancing or solubilising agent canbe added or the solvent can contain 10-60% w/w of propylene glycol orthe like.

The pharmaceutical compositions can contain one or more pharmaceuticallyacceptable sterile isotonic aqueous or nonaqueous solutions,dispersions, suspensions or emulsions, or sterile powders, which can bereconstituted into sterile injectable solutions or dispersions justprior to use. Such pharmaceutical compositions can contain antioxidants;buffers; bacteriostats; solutes, which render the formulation isotonicwith the blood of the intended recipient; suspending agents; thickeningagents; preservatives; and the like.

Examples of suitable aqueous and nonaqueous carriers, which can beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

In some embodiments, in order to prolong the effect of an activeingredient, it is desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This can be accomplished by theuse of a liquid suspension of crystalline or amorphous material havingpoor water solubility. The rate of absorption of the active ingredientthen depends upon its rate of dissolution which, in turn, can dependupon crystal size and crystalline form. Alternatively, delayedabsorption of a parenterally-administered active ingredient isaccomplished by dissolving or suspending the compound in an oil vehicle.In addition, prolonged absorption of the injectable pharmaceutical formcan be brought about by the inclusion of agents that delay absorptionsuch as aluminum monostearate and gelatin.

Controlled release parenteral compositions can be in form of aqueoussuspensions, microspheres, microcapsules, magnetic microspheres, oilsolutions, oil suspensions, emulsions, or the active ingredient can beincorporated in biocompatible carrier(s), liposomes, nanoparticles,implants or infusion devices.

Materials for use in the preparation of microspheres and/ormicrocapsules include biodegradable/bioerodible polymers such aspolyglactin, poly-(isobutyl cyanoacrylate),poly(2-hydroxyethyl-L-glutamine) and poly(lactic acid).

Biocompatible carriers which can be used when formulating a controlledrelease parenteral formulation include carbohydrates such as dextrans,proteins such as albumin, lipoproteins or antibodies.

Materials for use in implants can be non-biodegradable, e.g.,polydimethylsiloxane, or biodegradable such as, e.g.,poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(orthoesters).

In embodiments, the active ingredient(s) are administered by aerosol.This is accomplished by preparing an aqueous aerosol, liposomalpreparation, or solid particles containing the compound. A nonaqueous(e.g., fluorocarbon propellant) suspension can be used. Thepharmaceutical composition can also be administered using a sonicnebulizer, which would minimize exposing the agent to shear, which canresult in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the active ingredient(s) together withconventional pharmaceutically-acceptable carriers and stabilizers. Thecarriers and stabilizers vary with the requirements of the particularcompound, but typically include nonionic surfactants (Tweens, Pluronics,or polyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

Dosage forms for topical or transdermal administration of an activeingredient(s) includes powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The activeingredient(s) can be mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants as appropriate.

Transdermal patches suitable for use in the present invention aredisclosed in Transdermal Drug Delivery: Developmental Issues andResearch Initiatives (Marcel Dekker Inc., 1989) and U.S. Pat. Nos.4,743,249, 4,906,169, 5,198,223, 4,816,540, 5,422,119, 5,023,084, whichare hereby incorporated by reference. The transdermal patch can also beany transdermal patch well-known in the art, including transscrotalpatches. Pharmaceutical compositions in such transdermal patches cancontain one or more absorption enhancers or skin permeation enhancerswell-known in the art (see, e.g., U.S. Pat. Nos. 4,379,454 and4,973,468, which are hereby incorporated by reference). Transdermaltherapeutic systems for use in the present invention can be based oniontophoresis, diffusion, or a combination of these two effects.

Transdermal patches have the added advantage of providing controlleddelivery of active ingredient(s) to the body. Such dosage forms can bemade by dissolving or dispersing the active ingredient(s) in a propermedium. Absorption enhancers can also be used to increase the flux ofthe active ingredient across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe active ingredient(s) in a polymer matrix or gel.

Such pharmaceutical compositions can be in the form of creams,ointments, lotions, liniments, gels, hydrogels, solutions, suspensions,sticks, sprays, pastes, plasters and other kinds of transdermal drugdelivery systems. The compositions can also include pharmaceuticallyacceptable carriers or excipients such as emulsifying agents,antioxidants, buffering agents, preservatives, humectants, penetrationenhancers, chelating agents, gel-forming agents, ointment bases,perfumes, and skin protective agents.

Examples of emulsifying agents include, but are not limited to,naturally occurring gums, e.g. gum acacia or gum tragacanth, naturallyoccurring phosphatides, e.g. soybean lecithin and sorbitan monooleatederivatives.

Examples of antioxidants include, but are not limited to, butylatedhydroxy anisole (BHA), ascorbic acid and derivatives thereof, tocopheroland derivatives thereof, and cysteine.

Examples of preservatives include, but are not limited to, parabens,such as methyl or propyl p-hydroxybenzoate and benzalkonium chloride.

Examples of humectants include, but are not limited to, glycerin,propylene glycol, sorbitol and urea.

Examples of penetration enhancers include, but are not limited to,propylene glycol, DMSO, triethanolamine, N,N-dimethylacetamide,N,N-dimethylformamide, 2-pyrrolidone and derivatives thereof,tetrahydrofurfuryl alcohol, propylene glycol, diethylene glycolmonoethyl or monomethyl ether with propylene glycol monolaurate ormethyl laurate, eucalyptol, lecithin, Transcutol, and Azone®.

Examples of chelating agents include, but are not limited to, sodiumEDTA, citric acid and phosphoric acid.

Examples of gel forming agents include, but are not limited to,Carbopol, cellulose derivatives, bentonite, alginates, gelatin andpolyvinylpyrrolidone.

In addition to the active ingredient(s), the ointments, pastes, creams,and gels of the present invention can contain excipients, such as animaland vegetable fats, oils, waxes, paraffins, starch, tragacanth,cellulose derivatives, polyethylene glycols, silicones, bentonites,silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain excipients such as lactose, talc, silicicacid, aluminum hydroxide, calcium silicates and polyamide powder, ormixtures of these substances. Sprays can additionally contain customarypropellants, such as chlorofluorohydrocarbons, and volatileunsubstituted hydrocarbons, such as butane and propane.

Injectable depot forms are made by forming microencapsule matrices ofcompound(s) of the invention in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of compound topolymer, and the nature of the particular polymer employed, the rate ofcompound release can be controlled. Examples of other biodegradablepolymers include poly(orthoesters) and poly(anhydrides). Depotinjectable formulations are also prepared by entrapping the drug inliposomes or microemulsions which are compatible with body tissue.

Subcutaneous implants are well-known in the art and are suitable for usein the present invention. Subcutaneous implantation methods arepreferably non-irritating and mechanically resilient. The implants canbe of matrix type, of reservoir type, or hybrids thereof. In matrix typedevices, the carrier material can be porous or non-porous, solid orsemi-solid, and permeable or impermeable to the active compound orcompounds. The carrier material can be biodegradable or may slowly erodeafter administration. In some instances, the matrix is non-degradablebut instead relies on the diffusion of the active compound through thematrix for the carrier material to degrade. Alternative subcutaneousimplant methods utilize reservoir devices where the active compound orcompounds are surrounded by a rate controlling membrane, e.g., amembrane independent of component concentration (possessing zero-orderkinetics). Devices consisting of a matrix surrounded by a ratecontrolling membrane also suitable for use.

Both reservoir and matrix type devices can contain materials such aspolydimethylsiloxane, such as Silastic™, or other silicone rubbers.Matrix materials can be insoluble polypropylene, polyethylene, polyvinylchloride, ethylvinyl acetate, polystyrene and polymethacrylate, as wellas glycerol esters of the glycerol palmitostearate, glycerol stearate,and glycerol behenate type. Materials can be hydrophobic or hydrophilicpolymers and optionally contain solubilising agents.

Subcutaneous implant devices can be slow-release capsules made with anysuitable polymer, e.g., as described in U.S. Pat. Nos. 5,035,891 and4,210,644, which are hereby incorporated by reference.

In general, at least four different approaches are applicable in orderto provide rate control over the release and transdermal permeation of adrug compound. These approaches are: membrane-moderated systems,adhesive diffusion-controlled systems, matrix dispersion-type systemsand microreservoir systems. It is appreciated that a controlled releasepercutaneous and/or topical composition can be obtained by using asuitable mixture of these approaches.

In a membrane-moderated system, the active ingredient is present in areservoir which is totally encapsulated in a shallow compartment moldedfrom a drug-impermeable laminate, such as a metallic plastic laminate,and a rate-controlling polymeric membrane such as a microporous or anon-porous polymeric membrane, e.g., ethylene-vinyl acetate copolymer.The active ingredient is released through the rate controlling polymericmembrane. In the drug reservoir, the active ingredient can either bedispersed in a solid polymer matrix or suspended in an unleachable,viscous liquid medium such as silicone fluid. On the external surface ofthe polymeric membrane, a thin layer of an adhesive polymer is appliedto achieve an intimate contact of the transdermal system with the skinsurface. The adhesive polymer is preferably a polymer which ishypoallergenic and compatible with the active drug substance.

In an adhesive diffusion-controlled system, a reservoir of the activeingredient is formed by directly dispersing the active ingredient in anadhesive polymer and then by, e.g., solvent casting, spreading theadhesive containing the active ingredient onto a flat sheet ofsubstantially drug-impermeable metallic plastic backing to form a thindrug reservoir layer.

A matrix dispersion-type system is characterized in that a reservoir ofthe active ingredient is formed by substantially homogeneouslydispersing the active ingredient in a hydrophilic or lipophilic polymermatrix. The drug-containing polymer is then molded into disc with asubstantially well-defined surface area and controlled thickness. Theadhesive polymer is spread along the circumference to form a strip ofadhesive around the disc.

A microreservoir system can be considered as a combination of thereservoir and matrix dispersion type systems. In this case, thereservoir of the active substance is formed by first suspending the drugsolids in an aqueous solution of water-soluble polymer and thendispersing the drug suspension in a lipophilic polymer to form amultiplicity of unleachable, microscopic spheres of drug reservoirs.

Any of the above-described controlled release, extended release, andsustained release compositions can be formulated to release the activeingredient in about 30 minutes to about 1 week, in about 30 minutes toabout 72 hours, in about 30 minutes to 24 hours, in about 30 minutes to12 hours, in about 30 minutes to 6 hours, in about 30 minutes to 4hours, and in about 3 hours to 10 hours. In embodiments, an effectiveconcentration of the active ingredient(s) is sustained in a subject for4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 24 hours, 48hours, 72 hours, or more after administration of the pharmaceuticalcompositions to the subject.

Dosages

When the agents described herein are administered as pharmaceuticals tohumans and animals, they can be given per se or as a pharmaceuticalcomposition containing active ingredient in combination with apharmaceutically acceptable carrier, excipient, or diluent.

Actual dosage levels and time course of administration of the activeingredients in the pharmaceutical compositions of the invention can bevaried so as to obtain an amount of the active ingredient which iseffective to achieve the desired therapeutic response for a particularpatient, composition, and mode of administration, without being toxic tothe patient. Generally, agents or pharmaceutical compositions of theinvention are administered in an amount sufficient to reduce oreliminate symptoms associated with influenza infection.

Exemplary dose ranges include 0.01 mg to 250 mg per day, 0.01 mg to 100mg per day, 1 mg to 100 mg per day, 10 mg to 100 mg per day, 1 mg to 10mg per day, and 0.01 mg to 10 mg per day. A preferred dose of an agentis the maximum that a patient can tolerate and not develop serious orunacceptable side effects. In embodiments, the agent is administered ata concentration of about 10 micrograms to about 100 mg per kilogram ofbody weight per day, about 0.1 to about 10 mg/kg per day, or about 1.0mg to about 10 mg/kg of body weight per day.

In embodiments, the pharmaceutical composition comprises an agent in anamount ranging between 1 and 10 mg, such as 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 mg.

In embodiments, the therapeutically effective dosage produces a serumconcentration of an agent of from about 0.1 ng/ml to about 50-100 μg/ml.The pharmaceutical compositions typically should provide a dosage offrom about 0.001 mg to about 2000 mg of compound per kilogram of bodyweight per day. For example, dosages for systemic administration to ahuman patient can range from 1-10 μg/kg, 20-80 μg/kg, 5-50 μg/kg, 75-150μg/kg, 100-500 μg/kg, 250-750 μg/kg, 500-1000 μg/kg, 1-10 mg/kg, 5-50mg/kg, 25-75 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 50-100 mg/kg, 250-500mg/kg, 500-750 mg/kg, 750-1000 mg/kg, 1000-1500 mg/kg, 1500-2000 mg/kg,5 mg/kg, 20 mg/kg, 50 mg/kg, 100 mg/kg, 500 mg/kg, 1000 mg/kg, 1500mg/kg, or 2000 mg/kg. Pharmaceutical dosage unit forms are prepared toprovide from about 1 mg to about 5000 mg, for example from about 100 toabout 2500 mg of the compound or a combination of essential ingredientsper dosage unit form.

In embodiments, about 50 nM to about 1 μM of an agent is administered toa subject. In related embodiments, about 50-100 nM, 50-250 nM, 100-500nM, 250-500 nM, 250-750 nM, 500-750 nM, 500 nM to 1 μM, or 750 nM to 1μM of an agent is administered to a subject.

Determination of an effective amount is well within the capability ofthose skilled in the art, especially in light of the detailed disclosureprovided herein. Generally, an efficacious or effective amount of anagent is determined by first administering a low dose of the agent(s)and then incrementally increasing the administered dose or dosages untila desired effect (e.g., reduced symptoms associated with influenzainfection) is observed in the treated subject, with minimal oracceptable toxic side effects. Applicable methods for determining anappropriate dose and dosing schedule for administration of apharmaceutical composition of the present invention are described, forexample, in Goodman and Gilman's The Pharmacological Basis ofTherapeutics, Goodman et al., eds., 11th Edition, McGraw-Hill 2005, andRemington: The Science and Practice of Pharmacy, 20th and 21st Editions,Gennaro and University of the Sciences in Philadelphia, Eds., LippencottWilliams & Wilkins (2003 and 2005), each of which is hereby incorporatedby reference.

Combination Therapies

The agents and pharmaceutical compositions described herein can also beadministered in combination with another therapeutic molecule. Thetherapeutic molecule can be any compound used to treat influenzainfection. Examples of such compounds include, but are not limited to,inhibitory nucleic acids that reduce influenza virus production,antiviral agents (e.g., amantadine, rimantadine, zanamivir, oseltamivir,and the like), toxins, and agents that reduce the symptoms associatedwith influenza infection (e.g., anti-inflammatories).

The influenza HA antibody can be administered before, during, or afteradministration of the additional therapeutic agent. In embodiments, theantibody is administered before the first administration of theadditional therapeutic agent. In embodiments, the antibody isadministered after the first administration of the additionaltherapeutic agent (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14days or more). In embodiments, the antibody is administeredsimultaneously with the first administration of the additionaltherapeutic agent.

The amount of therapeutic agent administered to a subject can readily bedetermined by the attending physician or veterinarian. Generally, anefficacious or effective amount of an antibody and an additionaltherapeutic is determined by first administering a low dose of one orboth active agents and then incrementally increasing the administereddose or dosages until a desired effect is observed (e.g., reducedinfluenza infection symptoms), with minimal or no toxic side effects.Applicable methods for determining an appropriate dose and dosingschedule for administration of a combination of the present inventionare described, for example, in Goodman and Gilman's The PharmacologicalBasis of Therapeutics, 11th Edition, supra, and in Remington: TheScience and Practice of Pharmacy, 20th and 21st Editions, supra.

Kits

The invention provides for kits for preventing or treating influenzainfection; neutralizing an influenza virus; inhibiting establishment ofinfluenza virus infection; inhibiting dissemination of influenza virusinfection; as well as inhibiting influenza virus entry into a cell. Inembodiments, the kit contains one or more agents or pharmaceuticalcompositions described herein. In embodiments, the kit providesinstructions for use. The instructions for use can pertain to any of themethods described herein. In related embodiments, the instructionspertain to using the agent(s) or pharmaceutical composition(s) fortreating or preventing influenza infection. Kits according to thisaspect of the invention may comprise a carrier means, such as a box,carton, tube or the like, having in close confinement therein one ormore container means, such as vials, tubes, ampules, bottles and thelike. In embodiments, the kit provides a notice in the form prescribedby a governmental agency regulating the manufacture, use, or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale of the kit and the componentstherein for human administration.

Examples

It should be appreciated that the invention should not be construed tobe limited to the examples that are now described; rather, the inventionshould be construed to include any and all applications provided hereinand all equivalent variations within the skill of the ordinary artisan.

Example 1: The Clonal Lineage of a Broadly Neutralizing Antibody

Rearranged Ig V_(H) and V_(L) genes were isolated by RT/PCR fromperipheral blood mononuclear cells, collected from a subject one weekafter vaccination with the 2007 trivalent inactivated vaccine (TIV)(Liao, H. X. et al. J. Virol. Methods 158:171-179 (2009)). Among theclonal lineages detected by sequencing the rearranged genes was thethree-member clone (mAbs CH65, CH66 and CH67) shown in FIG. 1A. Theinferred sequence of the unmutated common ancestor (UCA) of the clonallineage of antibodies CH65, CH66 and CH67 is unambiguous, except atposition 99 of the heavy chain, which might be either glycine oralanine. FIG. 1B shows an alignment of the amino acid sequences of eachantibody to the UCA. All three mature antibodies bind the H1hemagglutinin (HA) present in the vaccine (A/Solomon Islands/3/2006)with about equal affinity; the UCA binds much more weakly.

Example 2: Breadth of Neutralizing Activity

The heavy chain of CH65 differs from the UCA at 12 positions in thevariable domain; and at 6 positions its light chain. CH65 IgG1 and itsFab were expressed in 293T cells by transient transfection and purifiedas described below. Neutralization was tested against a large panel ofH1 isolates from the past 30 years, including vaccine strains from 1977,1991 and 1995, and observed strikingly broad potency (Table 1). CH67 wasalso tested against a subset of this panel (Table 1).

TABLE 1 Broad neutralization of seasonal influenza strains A/H1N1 byhuman MAb CH65 and CH67 mAb minimum effective concentration (ug/ml) H1N1virus strain CH65 CH67 A/USSR/90/1977* 100 25 A/Kawasaki/6/1986 0.0980.39 A/Texas/36/1991

Neg Neg A/Wellington/47/1992 Neg Neg A/Florida/2/1993 0.012 0.012A/Beijing/262/1995* 0.098 0.098 A/Shengzhen/227/1995 0.012 0.024A/Shanghai/8/1996 Neg Neg A/Johannesburg/159/1997 0.098 0.39A/Shanghai/2/1997 0.195 0.195 A/Moscow/13/1998 0.012 0.012A/Ostrava/801/1998 12.5 Neg A/New Caledonia/22/1999

0.391 0.195 A/Bangkok/163/2000 0.195 0.098 A/Fujian/156/2000 0.488 0.049A/Chile/8885/2001 0.195 0.098 A/Auckland/65/2001 0.195 0.098A/Neimenggu/52/2002^(#) 0.098 0.098 A/Brazil/1403/2003 3.125 0.195A/Canada/59/2004 0.098 0.098 A/Solomon Islands/03/2006*^(,+) 0.024 0.098A/Brisbane/59/2007

0.098 0.98 A/California/07/2009 (swine)* Neg 6.25 *Strains that wereincluded in seasonal vaccines.

H1 component of the vaccine received by the subject. ^(#)Originallyreported as insensitive to mAb CH65.

indicates data missing or illegible when filed

The antibody neutralized H1N1 strains isolated as early as 1986,covering 21 years of antigenic drift. As expected, it neutralizedA/Solomon Islands/3/2006, the H1 component of the 2007 vaccine. Of the36 strains tested, it failed to neutralize only six, including the 2009pandemic strain, A/Texas/36/1991 and A/USSR/90/1977. The CH67 antibodyhas a similar breadth; it also neutralizes (weakly) the 2009 pandemicstrain. There is also evidence that CH66 (and likely CH65) binds HA froman H3 virus (X31, a lab strain derived from the 1968 pandemic. Too fewHA-directed human monoclonal antibodies have been characterized forsystematic comparison, but neutralization by serum samples does notordinarily exhibit this degree of breadth.

Example 3: Structure of CH65:HA

A complex of the mAb CH65 Fab with the HA ectodomain from A/SolomonIslands/03/2006 (HA^(SI)) was crystallized, recorded diffraction to aminimum Bragg spacing of 3.2-Å (Table 2), and the structure wasdetermined by molecular replacement as outlined below.

TABLE 2 Crystallographic statistics Data collection Resolution (lastshell), Å 30.0-3.20 (3.31-3.20) Wavelength, Å 0.980 Space group I212121Unit cell dimensions (a, b, c), Å 155.0, 191.8, 332.1 Unit cell angles(α, β, γ), ° 90, 90, 90 I/σ (last shell) 16.7 (2.1)  Rsym (last shell),%  9.8 (63.7) Completeness (last shell), % 98.9 (94.4) Number ofrefections 368947 unique 80377 Redundancy 4.6 Refinement Resolution, Å30.0-3.20 Number of refections 80336 working 78354 free 1982 Rwork, %21.1 Rfree, % 24.8 Ramachandran plot, % 88.5/9.3/2.2(favored/additional/disallowed) Number of atoms: protein 21794 other(sulfate ions) 214 rmsd bond lengths, Å 0.010 rmsd bond angles, ° 1.287

The asymmetric unit of the crystal contains a single copy of the HAtrimer, with three bound Fabs (FIGS. 2A-2D). The final model includesall HA1 and HA2 residues in the expressed protein, except fourdisordered residues at the C-terminus of HA1. The electron density mapsshowed evidence for N-linked glycosylation at all eight potential siteson each monomer, and one or more sugar residues at five of thesepositions could be modeled. The Fab is well-ordered, except residue 1 ofthe light chain and residues 141-147 of the heavy chain; these residuesare all far from the binding site.

A/Solomon Islands/03/2006 (this work) and A/Puerto Rico/8/1934 (Gamblin,S. J. et al., Science 303:1838-1842 (2004)) are, to the inventors'knowledge, the only seasonal H1N1 strains for which a structure of theHA has been determined; others are either pandemic strains or animalinfluenza strains. Comparison, using the program DALI, shows thatHA^(SI) is similar to other H1N1 HAs, such as those of the pandemicisolates from 2009 (Cα RMSD 0.9 Å over 495 aligned residues, 79%sequence identity; PDB IDs 3LZG (Xu, R. et al., Science 328:357-360(2010)) and 3LYJ (Zhang, W. et al., Protein Cell 1:459-467 (2010))) and1918 (Cα RMSD 1.5 Å over 495 aligned residues, 85% sequence identity;PDB IDs 3LZF (Xu, R et al., Science 328:357-360 (2010)) and 1RUZ(Gamblin, S. J. et al., Science 303:1838-1842 (2004))) and the seasonalisolate from 1934 (Cα RMSD 1.5 Å over 482 aligned residues, 86% sequenceidentity; PDB ID 1RVZ (Gamblin, S. J. et al., Science 303:1838-1842(2004))). The vestigial esterase domain of HA^(SI) resembles that of the2009 HA more closely than it does those from the 1918 and 1934 HAs.

MAb CH65 binds the globular head of the HA trimer (FIGS. 1C and 2A). Theepitope includes both the receptor site and the antigenic sitedesignated Sb in an early analysis of H1 sequences (Caton, A. J. et al.,Cell 31:417-427 (1982)). The contact buries 858 Å² on the antibody and748 Å² on HA1. All three CDRs of the heavy chain, as well as CDR-L1 andL3 of the light chain, participate in the interface (FIGS. 1C and 2B-D).CDR-H3 inserts into the receptor site. Seven of its nineteen residuescontribute 402 Å² of buried surface area, or 47% of the completeinterface. The other CDRs form flanking interactions. CDR-L3 contactsthe N-terminal end of the short α-helix, site Sb, at the edge of thereceptor pocket, and CDR-H1 and -H2 contact a loop that protrudes fromHA1 adjacent to the C-terminus of the short α-helix.

Tables 3 and 4 summarize several of the critical interactions betweenthe antibody and HA.

TABLE 3 Residues in CDR H3 of CH65 that contact HA (Table disclosesresidues 104-107 of SEQ ID NO: 10) Arg104* Ser105 Val106 Asp107 Tyr109Tyr 110 Tyr 112 *for some influenza strains (not Solomon Islands),Arg104 might contact HA

TABLE 4 Residues in HA that contact CH65 CDR HA residue contacted CDR H1158 CDR H2 158-160 CDR H3 135-136; 190-195; 226 CDR L1 222; 225; 227 CDRL2 (none) CDR L3 187; 189

Example 4: CDR-H3 of mAb CH65 Compared to the Receptor

Because CDR-H3 inserts into the receptor site, this structure wascompared to that of the human receptor analog LSTc(sialic-acid-α2,6-galactose-β1,4-N-acteylglucosamine) bound to 1934 HA(PDB ID 1RVZ: reference (Gamblin, S. J. et al., Science 303:1838-1842(2004)))(FIGS. 3A and 3B). In CH65, Asp107 at the tip of CDR-H3 acceptshydrogen bonds from the backbone amide of HA1 Ala137, the side chainhydroxyl of Ser136, and the side chain Nε of Arg226. (Arginine is foundonly rarely at position 226: glutamine is more common. Arg226 adopts akinked conformation in the crystal structure; a glutamine would fitreadily, with its Nε in the same position as the corresponding atom ofthe arginine side chain.) The backbone amide of Val106 in the antibodydonates a hydrogen bond to the carboxyl oxygen of HA1 Val135 on HA1, andthe nonpolar side chain of Val106 is in van der Waals contact with HA1Trp153 and Leu194. In receptor analog LSTc, the carboxylate group ofsialic acid has the same contacts with HA1 as does the (chemicallyanalogous) side chain of Asp107, and the N-acetyl group interacts withHA in the same way as just described for the amide and side chain ofVal106. In short, mAb CH65 mimics most of the chemical groups on thehuman receptor that interact with HA.

Example 5: Glycosylation

Glycosylation at antigenic sites is an important mechanism of immuneevasion by influenza virus (Knossow, M. and Skehel, J. J. Immunology119:1-7 (2006); Wiley, D. C. and Skehel, J. J. Annu. Rev. Biochem.56:365-394 (1987); and Wei, C. J. et al., Sci. Transl. Med. 2:24ra21(2010)). In HA^(SI), glycosylation leaves sites Sb and Cb exposed,partially obscures site Ca, and entirely masks antigenic site Sa. SiteSa is the epitope recognized by antibody 2D1, the prototype forIg-mediated immunity to 2009 H1N1 in survivors of the 1918 epidemic (Xu,R. et al., Science 328:357-360 (2010)). Of the side chains in contactwith 2D1, 7/16 differ between HA^(SI) and 1918 HA; in comparison, only3/16 differ between 2009 pandemic HA and 1918 H A. Because the HA ofA/Solomon Islands/03/2006 is glycosylated at site Sa, neithervaccination with TIV-2007, nor prior infection with an A/SolomonIslands/03/2006-like strain could have elicited a 2D1-like immuneresponse.

Example 6: Affinity Maturation

The amino-acid sequence of CH65 is the result of affinity maturationfrom its UCA. Analysis of the structure in light of its clonal lineage(FIG. 1B) shows that the central interactions of the antibodies with HAhave remained unchanged by affinity maturation. The CDR-H3 has notmutated, nor has the contact of the light-chain CDR-L3 with theN-terminal end of the short α-helix, site Sb. (Ser93 of CDR-L3 is Asp inlineage member CH67; substitution to Asp may allow CH67 to accept ahydrogen-bond from HA Asn187.) Elsewhere on the interaction surface ofthe antibody, changes to two residues create additional hydrogen bondsbetween the antibody and HA^(SI). Light-chain residues Asp26 and Arg29in CDR-L1 have mutated from their respective germline counterparts, Asnand Ser. Asp26 accepts a hydrogen-bond from HA Lys222. Arg29 ispositioned to donate two hydrogen-bonds to HA Asp225. Other changes,including those at position 31 (Gly to Asp) and positions 33-35(Trp-Met-His to His-Ile-Asn) may exert subtle effects on theconformation of CDR-H3.

Example 7: CH65-CH67 Lineage Reactivity to Different Influenza Strains

The ability of CH65-CH67 to react with other influenza strains wasassessed. 293 T cells were transfected with full-length HA from strainX31 (H3 influenza strain) (FIGS. 4A-4C, top panel) or with cell-surfaceexpressed globular head from A/Solomon Islands/3/2006 (H1 influenzastrain) (FIGS. 4D-4F, bottom panel). Cells were fixed with formaldehydeand probed with CH65 Fab (FIGS. 4B and 4E) or CH66 full-length antibody(FIGS. 4C and 4F), followed by a FITC-conjugated secondary antibodyspecific for the human Fab. Cells were imaged by FITC emission (532 nm).As a control, transfected cells were probed with secondary antibody only(FIGS. 4A and 4D). These results indicate that the CH65-CH67 antibodies(e.g., CH66) also bind other influenza serotypes (e.g., H3).

As discussed in detail in the above examples, CH65 comes from an adultsubject in the US, who received the 2007 TIV one week before donating aplasma sample. It was assumed that the subject had been exposed to H1N1influenza strains in the past, so that the antibodies obtained byscreening with a panel of recombinant hemagglutinins (rHAs) were from asecondary response. Indeed, the number of mutations (overall frequencyabout 5%) is too great to have occurred within just one week of aprimary exposure. In the setting of TIV, a large fraction of thecirculating antibody secreting plasma cells one week post vaccinationare influenza specific (Wrammert, J. et al., Nature 453:667-671 (2008)).Unlike other methods (e.g., phage display) for high-throughput analysisof human B-cell responses, the procedure described herein to isolateCH65 detects paired rearranged V_(H) and V_(L) regions, and hencereconstructs the complete antigen combining site of the native antibody(Liao, H. X. et al. J. Virol. Methods 158:171-179 (2009)).

The CH65 antibody belongs to a relatively small clonal lineage ofdetected sequences, but there were presumably other members notrepresented among the expressed antibodies. Because the plasma cell fromwhich it came probably derived from a vaccine-stimulated memory cell,most of the mutations that separate it from the UA probably occurredduring the earlier primary response. The breadth of infectivityneutralization by CH65 implies that it might have arisen during nearlyany of the seasonal outbreaks of the two decades preceding 2007, as onlya small number of mutations during the secondary response could haveproduced a very tightly binding antibody from one of somewhat loweraffinity.

The antigen combining site of CH65 has no markedly atyptical structuralfeatures. It has contributions from V_(H) 1˜2, D_(H) 1˜1, and J_(H) 6and from Vκ3-˜21 and Jκ2 (Table 5).

TABLE 5 Gene usage in CH65 VH VL CDR3 CDR3 Mutation length No. Mutationlength No. ID V D J frequency of a.a Isotype ID V J frequency of a.aH0082 1~2 1~1 6 5.0% 19 G1 L0024 3~21 2 4.2% 11 H1226 1~2 1~1 6 4.8% 19G1 L0408 3~21 2 4.3% 11 H2250 1~2 1~1 6 4.7% 19 G1 L0797 3~21 2 3.3% 11

The 19-residue heavy-chain CDR3 is of roughly average length (Volpe, J.M. and Kepler, T. B., Immunome Res. 4:3 (2008)). Its sequence in themature antibody is the same as in the UCA. The VDJ recombination thatgave rise to the coding sequence of the UCA included 17 n-nucleotides(FIG. 5), so that six of the fourteen CDR3 residues are encoded by therandom insertions produced by imprecise joining. These six residuesinclude Val106 and Asp107, which together make the most criticalcontacts within the sialic-acid pocket. The predicted n-nucleotideadditions are somewhat fewer than average at the V-D junction andsomewhat greater than average at the D-J junction (Volpe, J. M. andKepler, T. B., Immunome Res. 4:3 (2008)).

The tip of the CH65 heavy-chain CDR3 is a strikingly faithful mimic ofthe sialic-acid surface that contacts HA. Early work on influenza virusantigenic variation led to discussion of an apparent conflict betweenescape from neutralization and conservation of an exposed receptorbinding site. The HA structure resolved the issue, by showing that thesialic-acid binding site is smaller than the footprint of a typicalantibody and hence that mutations in the periphery of the receptorpocket can interfere with neutralization without blocking receptorattachment (Wiley, D. C. et al., Nature 289:373-378 (1981); and Wilson,I. A. et al., Nature 289:366-373 (1981)). Indeed, variations affectingthe susceptibility to neutralization by Ab CH65 map to sites that flankthe receptor pocket but avoid any direct receptor contacts.

Two published structures of murine mAbs bound with H3 HAs show somedegree of penetration into the receptor site—in both cases, by theheavy-chain CDR3. Neither mAb mimics the sialic-acid interaction asextensively as does CH65. In one (PDB ID 1KEN (Barbey-Martin, C. et al.,Virology 294:70-74 (2002))), an aspartic acid side chain approaches thelocation of the sialic-acid carboxylate, but in an orientation that canaccept a hydrogen bond only from the hydroxyl of Ser136 and not from themain-chain NH of Asn 137. In the other (2VIR (Fleury, D. et al., Nat.Struct. Biol. 5:119-123 (1998))), a Tyr-Asp pair at the tip of the CDR3has an orientation related to that of the Val-Asp pair in our CH65:HAcomplex, and the aspartic acid side chain has the same hydrogen-bondingpattern, but the mimicry does not extend to any of the interactions ofthe receptor N-acetyl group. H3 HAs have leucine, rather than glutamineor arginine at position 226, so that additional polar contact is notavailable.

Sites of mutations in naturally occurring, seasonal antigenic variantsof HA are largely on the outward facing surface of HA1. Some relativelyrare antibodies that bind a conserved site along the “stem” of the HAhave come from phage-displayed libraries of unrelated, rearranged humanV_(H) genes (all from V_(H)1-69). The structure and characteristics ofCH65 show that it is also possible to elicit broadly neutralizing,receptor-binding site antibodies. A parallel can be drawn with thebroadly neutralizing, receptor-site antibodies against HIV-1 (e.g.,antibody VRC01), which are reasonably close mimics of the functionalreceptor, CD4 (Zhou T. et al., Science 329:811-817 (2010)).

An immunogen with an enhanced probability of eliciting a CH65-likeresponse may protect against series of seasonal strains. A strategy fordesigning such an immunogen, based on analysis of both the structure andthe lineage, could include (in addition to the native HA) a component toinduce a UCA-like primary response. Inspection of the differencesbetween CH65 and its UCA suggests that the principal changes affectingaffinity are in the light-chain CDR1, where mutations at positions 26and 29 have introduced salt bridges with HA (Table 6).

TABLE 6 Potential influence of residues in CH65 that have changed fromUCA CH65 UCA potential effect Heavy chain E1 Q distant from contact D31G near a contact, but no salt bridge or strong polar H-bond H33 Y noobvious likely perturbation I 34 M ″ N35 H ″ H52 N might compensate forY->H at 33 D57 G no obvious likely perturbation A75 S distant V83 L ″N84 S ″ G85 R ″ K87 R ″ Light chain D26 N adds salt bridge R29 S addssalt bridge N35 Y might affect hc:lc interface C48 Y changes at 48 and49 would compensate for ech other Y49 D ″ I 98 V distant

A modified HA, in which the same contacts instead gain stability frommutations in the antigen, might have the desired properties.

The lack of common resistance mutations among the many strains testedsuggests that Ab CH65 will be a useful template for a therapeuticantibody. Oseltamivir-resistant H1N1, which emerged rapidly beginning in2007-8, has become the predominant strain of seasonal influenza, andmanagement of severe infection could benefit from a broadly reacting,immune-based therapeutic (Dharan, N. J. et al. JAMA 301:1034-1041(2009)). Previous studies with a human mAb targeting the globular headof H5N1 indicate that the effective neutralizing concentrations for CH65will be protective in vivo (Simmons, C. P. et al. PLoS Med. 4:e178(2007)).

Accordingly, described herein are novel influenza HA antibodies thatwill be extremely effective in treating and preventing influenzainfection. As seasonal antigenic drift of circulating influenza virusleads to a requirement for frequent changes in vaccine composition,because exposure or vaccination elicits human antibodies with limitedcross-neutralization of drifted strains, there is a significant unmetneed for an effective therapy that can broadly neutralize influenzadrifted strains. The above results clearly demonstrate that use of thenovel antibodies provides a solution to this unmet need. Therapy withthe novel antibodies described herein is therefore a significant advancein the treatment of patients suffering from influenza infection.

The results reported herein were obtained using the following methodsand materials.

Clinical Sample

MAbs CH65, CH66 and CH67 were obtained from a subject vaccinated withthe 2007 TIV under a Duke Institutional Board approved human subjectsprotocol. The subject received the 2007-2008 Fluzone° (Sanofi Pasteur,Swiftwater, Pa.), which contained A/Solomon Islands/3/2006(H1N1),A/Wisconsin/67/2005(H3N2), and B/Malaysia/2506/2004. Blood was drawn onday 7 post-vaccination, and PBMC were isolated and cryopreserved on thesame day. Single plasmablasts were sorted into 96-well plates, using apanel of antibodies as described (Moody M A, et al., PLoS One 6:e25797(2011)). Single-cell RT/PCR was carried out to obtain DNA for sequencing(Liao, H. X. et al. J. Virol. Methods 158:171-179 (2009)), which wasdone in both forward and reverse directions using a BigDye® sequencingkit on an ABI 3700 (Ewing, B. et al., Genome Res. 8:175-185 (1998)) andassembled with a method based on quality scores at each position(Kepler, T. B. et al. BMC Genomics 11:444 (2010)). Ig isotype wasdetermined by local alignment with known sequences; V, D, and J regiongenes, CDR3 loop lengths, and mutation frequencies were determined bycomparison with the inferred unmutated ancestor.

Lineage Analysis

The UCA was inferred using a Bayesian method by first determining theclonal tree by maximum likelihood using DNAML (Felsenstein, J. J. Mol.Evol. 17:368-376 (1981)), and then computing the posterior jointdistribution on gene segments and recombination sites, conditional onthe inferred ML tree. The posterior probability mass function onnucleotides at each position was then obtained directly.

Expression and Purification of IgG and Fab

The variable regions of immunoglobulin heavy- and light-chain genes wereisolated by RT/PCR from single plasma cells as described above (Liao, H.X. et al. J. Virol. Methods 158:171-179 (2009)). For production ofpurified full-length IgG antibody, the V_(H) and Vκ genes of CH65, CH66and CH67 were cloned into a pcDNA 3.1 expression vector containingeither the human IgG1 constant region gene or the κ-chain constantregion gene (Liao, H. X. et al. J. Virol. Methods 158:171-179 (2009)).To produce the CH65 Fab, a 5′ primer, HV13274-F1(5′-AAGCTTACCATGCCGATGGGCTCC-3′ (SEQ ID NO: 47)), was designed tocontain a restriction site (Hind III) and sequences to anneal to the 5′sequences of the Ig signal peptide, and a 3′ primer, HV13221H-R474(5′-GAGCCCAAATCTTGTGACAAATGATCTAGA-3′ (SEQ ID NO: 48)) was designed tocontain a restriction site (XbaI) and to introduce a stop codon afterthe sequence (5′TCTTGTGACAAA3′ (SEQ ID NO: 49)), encoding amino acidresidues, SCDK (SEQ ID NO: 50), just before the hinge of the human IgG1constant region. PCR amplification, using these primers and thefull-length IgG1 heavy chain gene as template, yielded the Fab gene,which was cloned into pcDNA3.1/hygro (Nicely, N. I. et al., Nat. Struct.Mol. Biol. 17:1492-1494 (2010)). Recombinant, intact, CH65 IgG1 and itsFab were produced in 293T cells by co-transfection with the genesencoding heavy and light chains. The intact antibody was purified usinganti-human IgG beads (Sigma, St. Louis, Mo.); the Fab, using anti-Lchain beads (Sigma, St. Louis, Mo.) followed by FPLC gel filtration(Liao, H. X. et al. J. Virol. Methods 158:171-179 (2009); and Nicely, N.I. et al., Nat. Struct. Mol. Biol. 17:1492-1494 (2010)).

Infectivity Neutralization

Infectivity neutralization was analyzed in a microneutralization assaybased on the methods of the influenza reference laboratories at theCenters for Disease Control and Prevention (CDC) (Hancock, K. et al., N.Engl. J. Med. 361:1945-1952 (2009)). H1N1 historical virus stocks wereprovided by Vladimir Lugovtsev (Div. of Viral Products, CBER, FDA). Allviruses were titrated on MDCK cells and used at 100 TCID₅₀ per well (intriplicate). Two-fold serial dilutions of mAb CH65, starting at 100μg/ml, were mixed with virus stocks before addition to MDCK cellmonolayers. The minimum concentrations that completely inhibited virusreplication (EC₉₉) are reported in Table 1.

HA Expression and Purification

Codon-optimized cDNA of the ectodomain of HA A/Solomon Islands/03/2006was synthesized by GeneArt and subcloned into a pET vector modified forligation-independent cloning (LIC). The synthetic gene encoded asecretion signal at the N terminus, and, in place of the transmembranedomain, a thrombin cleavage site, a T4-fibritin “foldon” to promoteproper trimerization, and a His6 tag (SEQ ID NO: 46) at the C terminus.Trichoplusia ni (Hi-5) cells were infected with recombinant baculovirus.The supernatant was harvested at 48 hours post-infection bycentrifugation, concentrated and diafiltered against phosphate-bufferedsaline with 40 mM imidazole, and loaded onto Ni-NTA resin. The proteinwas eluted, dialyzed, and incubated overnight with TPCK-treated trypsinat 1:500 mass ratio to remove the trimerization and His6 tags (SEQ IDNO: 46) and to cleave the HA0 precursor peptide. The protein was furtherpurified by gel filtration chromatography on Superdex 200 (GEHealthcare).

Crystallization

The CH65 Fab and the A/Solomon Islands/03/2006 HA were incubated in4.5:1 molar ratio, and the resulting 3:1 complex was separated fromexcess Fab by gel filtration chromatography on Superdex 200 in 10 mMHepes pH 7.5, 150 mM NaCl. The complex was concentrated to an absorbanceof 10 at 280 nm (approximately 6 mg/mL). Crystals were grown in hangingdrops over a reservoir containing 2.2 M ammonium sulfate, 100 mM Tris pH7.5, and 5% PEG-400 at 18 degrees C. Crystallization was improved bymicroseeding. After 3-14 days, crystals were cryoprotected by addingreservoir solution supplemented with 15% glycerol to the drop, thenharvested and flash cooled in liquid nitrogen.

Structure Determination and Refinement

Diffraction experiments were performed at beamline 24-ID-E at theAdvanced Photon Source. A dataset at 3.2-Å resolution was collected froma single ˜50×50×300 μm rod and processed using HKL2000 (Table 2).Molecular replacement (MR) calculations were performed with PHASER(McCoy, A. J. et al., J. Appl. Crystallogr. 40:658-674 (2007)), using1934 H1 HA (PDB ID 1RVZ (Gamblin, S. J. et al., Science 303:1838-1842(2004))) as the starting model. Initial phases from MR enabled a searchfor Fab molecules by phased molecular replacement in MOLREP, using alibrary of Fab structures. The model was refined in CNS (Brunger, A. T.,Nat. Protoc. 2:2728-2733 (2007)) by simulated annealing using deformableelastic network restraints and rebuilt in COOT (Emsley, P. and Cowtan,K., Acta Crystallogr. D. Biol. Crystallogr. 60:2126-2132 (2004)).N-linked glycans from a high-resolution structure were fitted intoexperimental electron density maps where appropriate. Strong 3-foldnon-crystallographic symmetry restraints were applied to HA and to eachdomain of the Fab throughout refinement, allowing variation in the anglebetween the conserved domain and the variable domain of the Fab.Finally, 3 cycles of individual positional and B-factor refinement inPHENIX (Adams, P. D. et al., Acta Crystallogr. D. Biol. Crystallogr.58:1948-1954 (2002)) resulted in a model in good agreement with observedintensities (R/Rfree=21.1/24.8%) (Table 2). Coordinates and diffractiondata have been submitted to the PDB, accession number 3SM5.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

INCORPORATION BY REFERENCE

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

1.-33. (canceled)
 34. A method of treating or preventing an influenzavirus infection in a subject in need thereof, wherein the methodcomprises administering to the subject an effective amount of i) ananti-influenza antibody or antibody fragment that specifically bindsinfluenza hemagglutinin (HA) at residues 136, 137 and 226 of the HApolypeptide sequence and thereby reduces or inhibits influenzahemagglutinin binding to sialic acid, wherein the anti-influenzaantibody or antibody fragment comprises at least one sequence selectedfrom the group consisting of: a variable heavy (V_(H)) chain sequenceSEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or one ormore heavy chain CDR regions present in a variable heavy (V_(H)) chainamino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, orSEQ ID NO: 12; and a variable light (V_(L)) chain sequence SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or one or more lightchain CDR regions present in a variable light (V_(L)) chain amino acidsequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO:16 or ii) a pharmaceutical composition comprising said anti-influenzaantibody or antibody fragment, thereby treating or preventing influenzavirus infection in the subject. 35.-38. (canceled)
 39. The method ofclaim 34, wherein the influenza is H1N1, H2N2, H3N2, or a human adaptedH5 strain.
 40. The method of claim 34, wherein the subject has or is atrisk of developing an influenza infection.
 41. The method of claim 40,wherein the subject is a mammal.
 42. The method of claim 41, wherein thesubject is human.
 43. The method of claim 40, wherein the subject issusceptible to viral infection.
 44. The method of claim 43, wherein thesubject is a pregnant female.
 45. The method of claim 43, wherein thesubject is a young subject or an infant subject.
 46. The method of claim43, wherein the subject is an elderly subject.
 47. The method of claim34, wherein i) the anti-influenza antibody or antibody fragment, or ii)the pharmaceutical composition is administered by intramuscularinjection, intravenous injection, subcutaneous injection, or inhalation.48.-58. (canceled)
 59. An anti-influenza Fab that specifically bindsinfluenza hemagglutinin (HA) at residues 136, 137 and 226 of the HApolypeptide sequence and thereby reduces or inhibits influenzahemagglutinin binding to sialic acid.
 60. The anti-influenza Fab ofclaim 59, wherein the HA polypeptide sequence is selected from the groupconsisting of SEQ ID NOS: 17-44.
 61. The anti-influenza Fab of claim 59,wherein the HA is selected from the group consisting of H1 HA, H2 HA, H3HA, or H5 HA.
 62. The anti-influenza Fab of claim 61, wherein the Fabfurther binds HA at one or more HA residues selected from the groupconsisting of: HA residues 158-160; HA residues 190-195; HA residues222, 225, and 227; and A/Solomon Islands/3/2006 HA residues 187 and 189.63. The anti-influenza Fab of claim 62, wherein the Fab further contactsan HA residue selected from the group consisting of HA residues 190-195.64. The anti-influenza Fab of claim 61, wherein the antibodycomplementary determining region (CDR) H1 region contacts HA residue158; the CDR H2 region contacts HA residues 158-160; the CDR H3 regioncontacts HA residues 135-136, 190-194 and 226; the CDR L1 regioncontacts HA residues 222, 225 and 227; or the CDR L3 region contacts HAresidues 187 and 198.